Thanks to Brad Lackford for assistance optimizing some of the techniques described in this manuscript.

1. Entretien de lignées cellulaires de macrophages <ol><li> Cultiver RAW264.7 et des lignées de cellules J774A.1 dans du DMEM supplémenté avec 10% de sérum bovin fœtal (FBS) à 37 ° C en présence de 5% de CO 2. Pour aider à maintenir la stérilité, ajouter 1% de pénicilline / streptomycine (pénicilline / streptomycine) aux médias (bien que ce ne soit pas strictement nécessaire). </li><li> Étape critique: Veiller à ce que les macrophages sont de plus en plus dans un état ​​sain pour la transfection efficace et knockdown gène siRNA induite. Utiliser des cellules entre passages 3 et 10 après décongélation frais, comme ces lignes de macrophages présentent la meilleure efficacité de la transfection et maintenir robuste survie post-transfection à ce stade; ne pas utiliser un numéro de passage plus élevé (&gt; 20), que l&#39;efficacité de la transfection et gène Knockdown fois diminuent considérablement. </li><li> Pour optimiser la transfection de siRNA, les cellules des plaques à 20% de confluence et les poussent un ou deux jours jusqu&#39;à ce que les cellules atteignent pas plus de 80% de confluence. Cellules envahies ne transfecter efficiently. </li></ol> 2. Programmation du système de navette à 96 puits pour la transfection <ol><li> Lancez le logiciel de navette de 96 puits sur l&#39;ordinateur connecté à la navette. Sélectionnez nouveau fichier de paramètres dans le menu en haut à gauche du fichier. </li><li> Mettez en surbrillance les puits qui seront transfectées sur le affichée plaque de 96 puits schématique. Une boîte verte indique que les puits sont sélectionnés. </li><li> Sélectionnez le programme qui correspond à la lignée cellulaire de macrophages utilisé (DS-136 pour RAW264.7 ou DS-130 pour J774A.1) et sélectionnez Appliquer, située en dessous de la plaque de 96 puits schématisé. Notez que, après la sélection d&#39;un programme, les puits de la plaque schématique seront colorés en bleu foncé. Cercles vides indiquent puits qui ne sont pas attribués un programme et ne seront pas choqués. </li></ol> 3. Préparation des réactifs pour la transfection <ol><li> Préparer la solution de travail Nucleofector ™ en mélangeant SF lignée cellulaire de 96 puits solution Nucleofector ™avec la totalité du contenu du tube en supplément joint. Laisser les réactifs mixtes à équilibrer à température ambiante avant de l&#39;utiliser. Stocker l&#39;excès de solution complète Nucleofector ™ à 4 ° C pendant 3 mois pour une utilisation future. </li><li> Préchauffer la solution saline DMEM, RPMI-1640, 0,25% de trypsine / EDTA, et tamponnée au phosphate (PBS) à 37 ° C avant de préparer les macrophages pour la transfection. </li><li> Préparer le siRNA pour la transfection par la fonte de la glace. Afin de minimiser le nombre des dégels gel du siRNA, geler de nouveaux siRNA en aliquots la première fois qu&#39;ils sont utilisés. </li><li> Dans les premières expériences, utiliser siRNA à une dose relativement élevée de 2 pm (01:10 dilué de 20 pm stocks). Titrer la suite siRNA gène qui induisent robuste démontable vers le bas à des doses plus faibles, bien que des doses plus élevées sont nécessaires en général pour effet de choc efficace de gènes dans des macrophages par rapport à d&#39;autres types de cellules. NOTE: siRNA sont disponibles à partir de nombreuses sources; assez robuste knockdown de gène peut être obtenuutilisant SmartPools de siGenome, qui sont les piscines de quatre siRNA ciblant chaque gène. Comme témoin négatif, utiliser l&#39;une des diverses piscines non-ciblage siRNA ou siRNA. </li><li> Pour contrôler l&#39;efficacité de transfection, transfecter soit le plasmide contrôle pmaxGFP (fourni dans le kit Amaxa) ou siRNA marqués par fluorescence en parallèle avec les siRNA expérimentales. Expect 30 à 50% de la transfection de plasmide GFP (analysé par cytométrie en flux ou microscopie jours après la transfection) et&gt; 99% de transfection de siRNA marqué par fluorescence (analysé par cytométrie de flux, immédiatement après la transfection et la récupération). </li></ol> 4. Préparer les macrophages pour la transfection <ol><li> Détachez les macrophages adhérents des boîtes de culture cellulaire par traitement à la trypsine. Aspirer les médias et laver les cellules deux fois avec 10 ml de PBS. Après avoir enlevé le PBS, incuber les cellules avec préchauffé 0,25% de trypsine / EDTA (1 ml / 10 cm plaque) pendant 1-2 min jusqu&#39;à ce que les cellules commencent à se détacher. Tapoter légèrement la plaque et déplacer la solution lors de l&#39;incubation de maximiser la trypsine. </li><li> Après l&#39;incubation, ajouter 9 ml de milieu DMEM + FBS, mélanger doucement de haut en bas, et de recueillir les cellules par pipetage dans un tube stérile. </li><li> Comptez le nombre de macrophages isolés à l&#39;aide d&#39;un hémocytomètre. Attendez environ 10 millions de macrophages par 10 cm plaque. Calculer le nombre total de macrophages nécessaires; chaque puits sur la navette 96 puits nécessite 200 000 cellules. Pensez à ajouter la valeur des cellules de quelques puits supplémentaires de cas de pertes de pipetage. </li><li> Pipeter le nombre calculé de cellules dans un tube de centrifugation. Centrifuger les cellules pendant 10 min à 150 x g à température ambiante. Ne pas centrifuger trop rapidement car cela diminue la santé des macrophages et l&#39;efficacité de la transfection. </li></ol> 5. nucléofection de macrophages avec siRNA <ol><li> L&#39;utilisation d&#39;un plaque de 96 puits à fond rond stérile (qui facilite le mélange tout en minimisant la perte de réactif), ajouter 2 pi de chaque siRNA à chaque puits pour être transfète. Remarque: En fonction du nombre de siRNA, ceci peut être réalisé au cours de l&#39;étape de centrifugation des macrophages. </li><li> Aspirer les médias à partir des cellules centrifugés. Resuspendre doucement les cellules de solution Nucleofector ™ SF (contenant la solution de complément), en utilisant 20 pi par 200 000 cellules sédimentées. Une fois remis en suspension, utiliser les cellules immédiatement pour la transfection optimale. </li><li> Transfert cellules remises en suspension dans une cuve stérile; à l&#39;aide d&#39;une pipette multicanaux, le transfert de 20 pl du mélange de cellules remis en suspension dans chaque puits de la plaque à 96 puits contenant le siRNA. Mélanger doucement par aspiration et refoulement. </li><li> Transférer 20 pl du mélange de cellules / siRNA dans la plaque à 96 puits Nucleocuvette ™ étape critique:. Éviter de générer des bulles lors de l&#39;étape de transfert, car cela peut provoquer des erreurs durant nucléofection. </li><li> Après avoir placé tous les échantillons expérimentaux dans la plaque de transfection, couvrir la plaque de 96 puits avec le couvercle fourni, very touchez doucement sur une surface dure pour éliminer les bulles à partir des échantillons. </li><li> Placer la plaque de 96 puits avec couvercle dans le 96 puits navette Nucleofector et sélectionnez «télécharger et démarrer &#39;en bas à droite de l&#39;écran du programme. Avant nucléofection, suivez l&#39;invite de programme pour enregistrer les résultats. Remarque: Durant le processus nucléofection, puits transfectées avec succès seront représentés sur le 96 bien schématique à l&#39;écran par un cercle vert avec un signe plus. Un cercle rouge avec un signe moins indique une erreur, probablement en raison de bulles ou mal pipetage le volume correct de réactifs. </li></ol> 6. Récupération et placage de macrophages <ol><li> Immédiatement après nucléofection, à l&#39;aide d&#39;une pipette multicanaux, ajouter 80 ul de pré-chauffé (37 ° C) RMP1-1640 médias à chaque puits. Ajouter les médias sur le côté de chaque puits, permettant aux médias de mélanger lentement avec les cellules transfectées, qui sont très délicat à ce stade. Ne pas ajouter les médias trop vite, comme cette volontédiminuer considérablement la viabilité. </li><li> Placer les plaques à 96 puits et transfectées avec des échantillons du milieu RPMI-1640 dans un incubateur à 37 ° C avec 5% de CO2 pendant 2 min; ce qui est essentiel pour permettre aux cellules de se rétablir avant une autre manipulation. </li><li> Retirer la plaque à 96 puits à partir de l&#39;incubateur et de les transférer avec précaution le mélange à partir de chaque puits dans une plaque de culture tissulaire à 96 puits en utilisant une pipette multicanaux. </li><li> En utilisant une pipette multicanaux, ajouter 100 ul de pré-chauffé (37 ° C) du milieu DMEM + FBS à chaque puits. </li><li> Incuber le 96 et le tissu plaque de culture avec des échantillons dans un incubateur à 37 ° C avec 5% de CO 2 pendant 24-36 heures (optimiser le moment exact de siRNA individuels, bien que cette plage de temps fonctionne généralement bien). </li></ol> 7. La stimulation des macrophages avec LPS <ol><li> Après le 24-36 h post-nucléofection incubation, préparer LPS (ou autre PAMP) dans un milieu frais. Préparer suffisamment milieu frais pour tous les puits (200 pi media / puits de la plaque de 96 puits et un peu plus de pipetage). Diluer le LPS à une concentration finale de 20 ng / ml dans du DMEM + FBS. </li><li> Médias aspirer délicatement de la plaque de 96 puits et ajouter les milieux frais contenant les LPS aux cellules. </li><li> Surveiller la réponse au LPS ou d&#39;autres PAMP à divers moments après la stimulation. 6 heures est suffisante pour générer une réponse de cytokine inflammatoire robuste pour des cytokines telles que l&#39;IL-6 ou TNF. </li></ol> 8. Suivi de la réponse immunitaire innée induite, Gene Knockdown, et la viabilité dans les macrophages <ol><li> Pour surveiller induite par le LPS production de cytokines, pipeter le surnageant des cellules dans une plaque de 96 puits de stockage. La production de cytokine peut être ensuite mesurée par ELISA. </li><li> Une fois que le surnageant est éliminé, surveiller la viabilité des cellules en utilisant le diacétate de fluorescéine (lorsqu&#39;il est clivé par des estérases cellulaires dans les cellules vivantes, ce composé est fluorescent). Identifier les siRNA ciblant les gènes qui contrôlent la viabilité des cellules en utilisant tson approche. Remarque: le processus de transfection lui-même devrait avoir peu d&#39;effet sur la viabilité si elle est effectuée correctement. <ol><li> Ajouter diacétate de fluorescéine (5 mg / ml dans de l&#39;acétone actions) à chaque puits (1: 100 dilution finale dans du PBS). </li><li> Incuber à température ambiante pendant une à cinq minutes, puis surveiller la fluorescence dans les cellules en utilisant un lecteur de plaque (460 nm d&#39;excitation, 515 nm émission). </li><li> Facultatif: Pour veiller à ce que la fluorescence dans les puits est uniforme, la lyse des cellules en utilisant des tampons tels que RIPA; cela fournit un signal fluorescent uniforme dans le puits, comme certains lecteurs de plaques sont configurés pour surveiller seulement une petite partie du puits; si les cellules ne sont pas étalées uniformément, le diacétate de fluorescéine pourrait donner une lecture inexacte. </li></ol></li><li> Comme alternative à la surveillance de la viabilité cellulaire, contrôler gène induite par le siRNA-knockdown par qPCR en utilisant l&#39;ARN provenant des cellules adhérentes. <ol><li> Après que le surnageant est séparé des cellules (étape 8.1 ci-dessus), annonced tampon RLT (qui est utilisé pour la lyse et la préservation de l&#39;ARN) directement aux cellules adhérentes de les lyser. </li><li> Isoler l&#39;ARN en utilisant un kit de préparation d&#39;ARN tels que le mini-kit RNeasy, et en utilisant qPCR avec des amorces spécifiques d&#39;un gène par rapport au gène surveiller démontable pour contrôler le traitement des siRNA. Utilisez amorces à Βactin ou GAPDH pour la normalisation. </li></ol></li><li> Comme autre alternative à surveiller d&#39;autres aspects de la réponse immunitaire innée, surveiller la capacité phagocytaire des macrophages par l&#39;addition marqué au FITC E. particules coli directement sur ​​les cellules adhérentes en utilisant un kit. Suivant le protocole du fabricant, qui ne prend que quelques heures, contrôler l&#39;absorption phagocytaire utilisant un lecteur de plaque ou microscopie. </li></ol>

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Les auteurs déclarent qu&#39;ils ont aucun intérêt financier concurrents. Cet article a été parrainé par Lonza, Inc.

De nombreuses études ont été publiées dans lequel les gènes individuels ont été pris pour cible par siRNA dans les macrophages murins. Bien que la transfection médiée par des lipides a été utilisé pour fournir des siRNA pour les lignées de cellules macrophages sur une base individuelle, ces méthodes souffrent des effets potentiels sur la viabilité, knock-down de gènes limitée, et la variabilité du gène à gène. Pour développer des tests plus robustes qui pourraient être utilisés pour cibler les g?...

Dans ce protocole, nous décrivons des méthodes efficaces pour inhiber des gènes dans des lignées cellulaires de macrophages de souris à l&#39;aide de siRNA et de surveiller les effets de ces traitements sur la réponse immunitaire innée. Ces procédures sont effectuées en format 96 puits, permettant écrans ARNi de la mode à moyen ou haut débit. En réponse à une infection, l&#39;homme une réponse immunitaire innée immédiate et une réponse immunitaire adaptative plus lente mais plus précise. Cette réponse immunitaire innée rapide comprend le recrutement et l&#39;activation des cellules immunitaires innées, y compris les macrophages phagocytaires 1. Macrophages activées de façon classique sont impliquées dans les réponses inflammatoires aiguës et produisent des protéines antimicrobiennes et composés, les cytokines et les chimiokines, et les antigènes présents 2,3. Macrophages activés alternativement jouent un rôle dans la régulation de l&#39;immunité, le maintien de la tolérance, la réparation des tissus, cicatrisation des plaies et 4-8. En raison de leur large éventail de fonctions, Les macrophages peuvent jouer un rôle dans de nombreuses maladies, y compris l&#39;athérosclérose, l&#39;arthrite, le cancer et 9. Ainsi, l&#39;étude des macrophages a été un élément clé de la recherche depuis un certain temps dans une grande variété de domaines des maladies. En dépit de leur importance dans la réponse immunitaire innée, les macrophages peut être difficile de travailler avec des cellules. En particulier, il est difficile d&#39;obtenir une transfection efficace en utilisant des réactifs lipidiques dans les macrophages sans toxicité associée 10,11. En outre, même lorsque le siRNA est rendu efficace à des macrophages, la robustesse de gène induite par ARNi démontable peut être assez souvent modérée et peut varier d&#39;un gène à gène. Pour remédier à ces difficultés techniques, nous avons optimisé la transfection et des techniques knockdown 12-16 dans deux lignées de cellules de type macrophage de souris, RAW264.7 17 et 18 J774A.1. Cette approche utilise la 96 puits système de navette Amaxa Nucleofector pour la transfection; ce systèmeutilise une combinaison de réactifs spécialisés et l&#39;électroporation pour transfecter des cellules dans un format à 96 puits 19. Après la transfection, on décrit des procédés pour optimiser la récupération et la viabilité cellulaire et de maximiser la suite gène induite par effet de choc de siRNA. Pour illustrer l&#39;utilité de cette approche, nous décrivons un protocole de délivrance d&#39;ARNi de ces lignées de cellules macrophages, stimuler ces cellules avec du lipopolysaccharide (LPS) et contrôler la réponse immunitaire innée à l&#39;échelle de la production de plusieurs cytokines pro-inflammatoires. Nous fournissons des données de l&#39;échantillon dans lequel nous ciblons la famille des récepteurs Toll-like (TLR), dont les membres sentir LPS et autres motifs moléculaires de pathogènes associés (PAMP), pour réguler l&#39;immunité innée.

<table border="0" cellpadding="0" cellspacing="0" >
	<tbody>
		<tr >
			<td >Amaxa nucleofector 96-well shuttle system</td>
			<td>Lonza</td>
			<td>AAM-1001S</td>
		<td></td></tr>
		<tr >
			<td >Amaxa SF cell line 96-well nucleofector kit</td>
			<td>Lonza</td>
			<td>V4SC-2096</td>
		<td></td></tr>
		<tr >
			<td >RAW264.7 mouse macrophage cell line</td>
			<td>ATCC</td>
			<td>TIB-71</td>
		<td></td></tr>
		<tr >
			<td >J774A.1 mouse macrophage cell line</td>
			<td>ATCC</td>
			<td>TIB-67</td>
		<td></td></tr>
		<tr >
			<td >siGenome smartpool siRNA</td>
			<td>Dharmacon</td>
			<td>varies depending on gene</td>
		<td></td></tr>
		<tr >
			<td >Non-targeting control siRNA pool</td>
			<td>Dharmacon</td>
			<td>D-001206-13-20</td>
		<td></td></tr>
		<tr >
			<td >Block-iT fluorescent oligo for electrooration</td>
			<td>Invitrogen</td>
			<td>13750062</td>
		<td></td></tr>
		<tr >
			<td >Ultrapure E. coli O111:B4 LPS</td>
			<td>List Biological Laboratories</td>
			<td>421</td>
		<td></td></tr>
		<tr >
			<td >DMEM, high glucose</td>
			<td>Invitrogen</td>
			<td>11995-065</td>
		<td></td></tr>
		<tr >
			<td >RPM1-1640</td>
			<td>Invitrogen</td>
			<td>11875-093</td>
		<td></td></tr>
		<tr >
			<td >Penicillin-Streptomycin Solution (Pen/Strep)</td>
			<td>Fisher</td>
			<td>SV30010</td>
		<td></td></tr>
		<tr >
			<td >0.25% Trypsin-EDTA</td>
			<td>Invitrogen</td>
			<td>25200072</td>
		<td></td></tr>
		<tr >
			<td >96 well tissue culture plates</td>
			<td>Fisher</td>
			<td>07-200-89 </td>
		<td></td></tr>
		<tr >
			<td >96 well round bottom sterile plates (not coated)</td>
			<td>Fisher</td>
			<td>07-200-745</td>
		<td></td></tr>
		<tr >
			<td >Mouse IL-6 Duoset ELISA kit</td>
			<td>R&#38;D Biosystems</td>
			<td>DY406</td>
		<td></td></tr>
		<tr >
			<td >Mouse TNFa Duoset ELISA kit</td>
			<td>R&#38;D Biosystems</td>
			<td>DY410</td>
		<td></td></tr>
		<tr >
			<td >Fluorescein diacetate</td>
			<td>Sigma-Aldrich</td>
			<td>F7378</td>
		<td></td></tr>
		<tr >
			<td >RLT Bufer</td>
			<td>Qiagen</td>
			<td>79216</td>
		<td></td></tr>
		<tr >
			<td >Rneasy mini kit</td>
			<td>Qiagen</td>
			<td>74134</td>
		<td></td></tr>
		<tr >
			<td >Vybrant Phagocytosis Assay Kit</td>
			<td>Invitrogen</td>
			<td>V-6694</td>
		<td></td></tr>
	</tbody>
</table>

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 pathogen, produce various cytokines and chemokines to recruit and stimulate other immune cells, and present antigens to stimulate the adaptive immune response. Thus, being able to efficiently manipulate macrophages with techniques such as RNA-interference (RNAi) is critical to our ability to investigate this important innate immune cell. However, macrophages can be technically challenging to transfect and can exhibit inefficient RNAi-induced gene knockdown. In this protocol, we describe methods to efficiently transfect two mouse macrophage cell lines (RAW264.7 and J774A.1) with siRNA using the Amaxa Nucleofector 96-well Shuttle System and describe procedures to maximize the effect of siRNA on gene knockdown. Moreover, the described methods are adapted to work in 96-well format, allowing for medium and high-throughput studies. To demonstrate the utility of this approach, we describe experiments that utilize RNAi to inhibit genes that regulate lipopolysaccharide (LPS)-induced cytokine production.

Pour démontrer l&#39;efficacité de la transfection en utilisant cette approche, nous avons suivi l&#39;absorption de FITC marqué siRNA l&#39;aide d&#39;un cytomètre de flux (Figure 1). Pour illustrer l&#39;utilité de notre approche pour le suivi de la réponse immunitaire innée, nous avons transfecté siRNA ciblant des gènes de régulation de l&#39;immunité innée connus dans la lignée cellulaire de macrophages RAW264.7, stimulé les cellules avec L...

Watch this Scientific Journal Video about Using RNA-interference to Investigate the Innate Immune Response in Mouse Macrophages at JoVE.com

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

In this protocol, we describe methods to efficiently transfect murine macrophage cell lines with siRNAs using the Amaxa Nucleofector 96-well Shuttle System, stimulate the macrophages with lipopolysaccharide, and monitor the effects on inflammatory cytokine production.

En utilisant l&#39;ARN interférence pour étudier la réponse immunitaire innée dans les macrophages de souris

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

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

Research

JoVE Journal

Immunology and Infection

10.6K vues.  National Jewish Health and University of Colorado School of Medicine. In this protocol, we describe methods to efficiently transfect murine macrophage cell lines with siRNAs using the Amaxa Nucleofector 96-well Shuttle System, stimulate the macrophages with lipopolysaccharide, and monitor the effects on inflammatory cytokine production.

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

RNA Interference in Ticks

Ticks are obligate hematophagous ectoparasites of wild and domestic animals and humans, and are considered to be second worldwide to mosquitoes as vectors of human diseases1 and the most important vectors affecting cattle industry worldwide2. Ticks are classified in the subclass Acari, order Parasitiformes, suborder Ixodida and are distributed worldwide from Arctic to tropical regions3. Despite efforts to control tick infestations, these ectoparasites remain a serious problem for human and animal health4,5.
RNA interference (RNAi)6 is a nucleic acid-based reverse genetic approach that involves disruption of gene expression in order to determine gene function or its effect on a metabolic pathway. Small interfering RNAs (siRNAs) are the effector molecules of the RNAi pathway that is initiated by double-stranded RNA (dsRNA) and results in a potent sequence-specific degradation of cytoplasmic mRNAs containing the same sequence as the dsRNA trigger7-9. Post-transcriptional gene silencing mechanisms initiated by dsRNA have been discovered in all eukaryotes studied thus far, and RNAi has been rapidly developed in a variety of organisms as a tool for functional genomics studies and other applications10.
RNAi has become the most widely used gene-silencing technique in ticks and other organisms where alternative approaches for genetic manipulation are not available or are unreliable5,11. The genetic characterization of ticks has been limited until the recent application of RNAi12,13. In the short time that RNAi has been available, it has proved to be a valuable tool for studying tick gene function, the characterization of the tick-pathogen interface and the screening and characterization of tick protective antigens14. Herein, a method for RNAi through injection of dsRNA into unfed ticks is described. It is likely that the knowledge gained from this experimental approach will contribute markedly to the understanding of basic biological systems and the development of vaccines to control tick infestations and prevent transmission of tick-borne pathogens15-19.

A method for RNA interference (RNAi) by injection of dsRNA into unfed ticks is described. RNAi is the most widely used gene-silencing technique in ticks where the use of other methods of genetic manipulation has been limited.

ARN interférence dans les tiques

Ticks are obligate hematophagous ectoparasites of wild and domestic animals and humans, and are considered to be second worldwide to mosquitoes as vectors ...

Recherche

Immunologie et infection

An Introduction to Parasitic Wasps of Drosophila and the Antiparasite Immune Response

Most known parasitoid wasp species attack the larval or pupal stages of Drosophila. While Trichopria drosophilae infect the pupal stages of the host (Fig. 1A-C), females of the genus Leptopilina (Fig. 1D, 1F, 1G) and Ganaspis (Fig. 1E) attack the larval stages. We use these parasites to study the molecular basis of a biological arms race. Parasitic wasps have tremendous value as biocontrol agents. Most of them carry virulence and other factors that modify host physiology and immunity. Analysis of Drosophila wasps is providing insights into how species-specific interactions shape the genetic structures of natural communities. These studies also serve as a model for understanding the hosts' immune physiology and how coordinated immune reactions are thwarted by this class of parasites.
The larval/pupal cuticle serves as the first line of defense. The wasp ovipositor is a sharp needle-like structure that efficiently delivers eggs into the host hemocoel. Oviposition is followed by a wound healing reaction at the cuticle (Fig. 1C, arrowheads). Some wasps can insert two or more eggs into the same host, although the development of only one egg succeeds. Supernumerary eggs or developing larvae are eliminated by a process that is not yet understood. These wasps are therefore referred to as solitary parasitoids.
Depending on the fly strain and the wasp species, the wasp egg has one of two fates. It is either encapsulated, so that its development is blocked (host emerges; Fig. 2 left); or the wasp egg hatches, develops, molts, and grows into an adult (wasp emerges; Fig. 2 right). L. heterotoma is one of the best-studied species of Drosophila parasitic wasps. It is a "generalist," which means that it can utilize most Drosophila species as hosts1. L. heterotoma and L. victoriae are sister species and they produce virus-like particles that actively interfere with the encapsulation response2. Unlike L. heterotoma, L. boulardi is a specialist parasite and the range of Drosophila species it utilizes is relatively limited1. Strains of L. boulardi also produce virus-like particles3 although they differ significantly in their ability to succeed on D. melanogaster1. Some of these L. boulardi strains are difficult to grow on D. melanogaster1 as the fly host frequently succeeds in encapsulating their eggs. Thus, it is important to have the knowledge of both partners in specific experimental protocols.
In addition to barrier tissues (cuticle, gut and trachea), Drosophila larvae have systemic cellular and humoral immune responses that arise from functions of blood cells and the fat body, respectively. Oviposition by L. boulardi activates both immune arms1,4. Blood cells are found in circulation, in sessile populations under the segmented cuticle, and in the lymph gland. The lymph gland is a small hematopoietic organ on the dorsal side of the larva. Clusters of hematopoietic cells, called lobes, are arranged segmentally in pairs along the dorsal vessel that runs along the anterior-posterior axis of the animal (Fig. 3A). The fat body is a large multifunctional organ (Fig. 3B). It secretes antimicrobial peptides in response to microbial and metazoan infections.
Wasp infection activates immune signaling (Fig. 4)4. At the cellular level, it triggers division and differentiation of blood cells. In self defense, aggregates and capsules develop in the hemocoel of infected animals (Fig. 5)5,6. Activated blood cells migrate toward the wasp egg (or wasp larva) and begin to form a capsule around it (Fig. 5A-F). Some blood cells aggregate to form nodules (Fig. 5G-H). Careful analysis reveals that wasp infection induces the anterior-most lymph gland lobes to disperse at their peripheries (Fig. 6C, D).
We present representative data with Toll signal transduction pathway components Dorsal and Sp&auml;tzle (Figs. 4,5,7), and its target Drosomycin (Fig. 6), to illustrate how specific changes in the lymph gland and hemocoel can be studied after wasp infection. The dissection protocols described here also yield the wasp eggs (or developing stages of wasps) from the host hemolymph (Fig. 8).

An Introduction to Parasitic Wasps of Drosophila and the Antiparasite Immune Response

Parasitoid (parasitic) wasps constitute a major class of natural enemies of many insects including Drosophila melanogaster. We will introduce the techniques to propagate these parasites in Drosophila spp. and demonstrate how to analyze their effects on immune tissues of Drosophila larvae.

Une introduction à guêpes parasites<em&gt; Drosophila</em&gt; Et la réponse immunitaire antiparasitaire

Most known parasitoid wasp species attack the larval or pupal stages of Drosophila. While Trichopria drosophilae infect the pupal stages of the host (Fig. ...

Intravital Imaging of the Mouse Thymus using 2-Photon Microscopy

Two-photon Microscopy (TPM) provides image acquisition in deep areas inside tissues and organs. In combination with the development of new stereotactic tools and surgical procedures, TPM becomes a powerful technique to identify "niches" inside organs and to document cellular "behaviors" in live animals. While intravital imaging provides information that best resembles the real cellular behavior inside the organ, it is both more laborious and technically demanding in terms of required equipment/procedures than alternative ex vivo imaging acquisition. Thus, we describe a surgical procedure and novel "stereotactic" organ holder that allows us to follow the movements of Foxp3+ cells within the thymus.
Foxp3 is the master regulator for the generation of regulatory T cells (Tregs). Moreover, these cells can be classified according to their origin: ie. thymus-differentiated Tregs are called "naturally-occurring Tregs" (nTregs), as opposed to peripherally-converted Tregs (pTregs). Although significant amount of research has been reported in the literature concerning the phenotype and physiology of these T cells, very little is known about their in vivo interactions with other cells. This deficiency may be due to the absence of techniques that would permit such observations. The protocol described in this paper provides a remedy for this situation.
Our protocol consists of using nude mice that lack an endogenous thymus since they have a punctual mutation in the DNA sequence that compromises the differentiation of some epithelial cells, including thymic epithelial cells. Nude mice were gamma-irradiated and reconstituted with bone marrows (BM) from Foxp3-KIgfp/gfp mice. After BM recovery (6 weeks), each animal received embryonic thymus transplantation inside the kidney capsule. After thymus acceptance (6 weeks), the animals were anesthetized; the kidney containing the transplanted thymus was exposed, fixed in our organ holder, and kept under physiological conditions for in vivo imaging by TPM. We have been using this approach to study the influence of drugs in the generation of regulatory T cells.

We have developed novel laboratory tools and protocols for intravital imaging acquisition of the thymus. Our technique should help in the identification of &#x201C;niches&#x201D; within the thymus where T cell development occurs.

Imagerie intravitale du Thymus souris utilisant la 2-microscopie biphotonique

Two-photon Microscopy (TPM) provides image acquisition in deep areas inside tissues and organs. In combination with the development of new stereotactic ...

Quantitative Measurement of the Immune Response and Sleep in Drosophila

A complex interaction between the immune response and host behavior has been described in a wide range of species. Excess sleep, in particular, is known to occur as a response to infection in mammals 1 and has also recently been described in Drosophila melanogaster2. It is generally accepted that sleep is beneficial to the host during an infection and that it is important for the maintenance of a robust immune system3,4. However, experimental evidence that supports this hypothesis is limited4, and the function of excess sleep during an immune response remains unclear. We have used a multidisciplinary approach to address this complex problem, and have conducted studies in the simple genetic model system, the fruitfly Drosophila melanogaster. We use a standard assay for measuring locomotor behavior and sleep in flies, and demonstrate how this assay is used to measure behavior in flies infected with a pathogenic strain of bacteria. This assay is also useful for monitoring the duration of survival in individual flies during an infection. Additional measures of immune function include the ability of flies to clear an infection and the activation of NF&kappa;B, a key transcription factor that is central to the innate immune response in Drosophila. Both survival outcome and bacterial clearance during infection together are indicators of resistance and tolerance to infection. Resistance refers to the ability of flies to clear an infection, while tolerance is defined as the ability of the host to limit damage from an infection and thereby survive despite high levels of pathogen within the system5. Real-time monitoring of NF&kappa;B activity during infection provides insight into a molecular mechanism of survival during infection. The use of Drosophila in these straightforward assays facilitates the genetic and molecular analyses of sleep and the immune response and how these two complex systems are reciprocally influenced.

Quantitative Measurement of the Immune Response and Sleep in Drosophila

To understand a link between the immune response and behavior, we describe a method to measure locomotor behavior in Drosophila during bacterial infection as well as the ability of flies to mount an immune response by monitoring survival, bacterial load, and real-time activity of a key regulator of innate immunity, NF&kappa;B.

Mesure quantitative de la réponse immunitaire et le sommeil en<em&gt; Drosophila</em

A complex interaction between the immune response and  host behavior has been described in a wide range of species. Excess sleep, in  particular, is known ...

Quantification of the Respiratory Burst Response as an Indicator of Innate Immune Health in Zebrafish

The phagocyte respiratory burst is part of the innate immune response to pathogen infection and involves the production of reactive oxygen species (ROS). ROS are toxic and function to kill phagocytized microorganisms. In vivo quantification of phagocyte-derived ROS provides information regarding an organism's ability to mount a robust innate immune response. Here we describe a protocol to quantify and compare ROS in whole zebrafish embryos upon chemical induction of the phagocyte respiratory burst. This method makes use of a non-fluorescent compound that becomes fluorescent upon oxidation by ROS. Individual zebrafish embryos are pipetted into the wells of a microplate and incubated in this fluorogenic substrate with or without a chemical inducer of the respiratory burst. Fluorescence in each well is quantified at desired time points using a microplate reader. Fluorescence readings are adjusted to eliminate background fluorescence and then compared using an unpaired t-test. This method allows for comparison of the respiratory burst potential of zebrafish embryos at different developmental stages and in response to experimental manipulations such as protein knockdown, overexpression, or treatment with pharmacological agents. This method can also be used to monitor the respiratory burst response in whole dissected kidneys or cell preparations from kidneys of adult zebrafish and some other fish species. We believe that the relative simplicity and adaptability of this protocol will complement existing protocols and will be of interest to researchers who seek to better understand the innate immune response.

The innate immune response protects organisms against pathogen infection. A critical component of the innate immune response, the phagocyte respiratory burst, generates reactive oxygen species that kill invading microorganisms. We describe a respiratory burst assay that quantifies reactive oxygen species produced when the innate immune response is chemically induced.

Quantification de la réponse du Rafale respiratoire comme indicateur d&#39;Innate Immune Health chez le poisson zèbre

The phagocyte respiratory burst is part of the innate immune response to  pathogen infection and involves the production of reactive oxygen species (ROS). ...

Dissecting Innate Immune Signaling in Viral Evasion of Cytokine Production

In response to a viral infection, the host innate immune response is activated to up-regulate gene expression and production of antiviral cytokines. Conversely, viruses have evolved intricate strategies to evade and exploit host immune signaling for survival and propagation. Viral immune evasion, entailing host defense and viral evasion, provides one of the most fascinating and dynamic interfaces to discern the host-virus interaction. These studies advance our understanding in innate immune regulation and pave our way to develop novel antiviral therapies.
Murine &#947;HV68 is a natural pathogen of murine rodents. &#947;HV68 infection of mice provides a tractable small animal model to examine the antiviral response to human KSHV and EBV of which perturbation of in vivo virus-host interactions is not applicable. Here we describe a protocol to determine the antiviral cytokine production. This protocol can be adapted to other viruses and signaling pathways.
Recently, we have discovered that &#947;HV68 hijacks MAVS and IKK&#946;, key innate immune signaling components downstream of the cytosolic RIG-I and MDA5, to abrogate NF&#922;B activation and antiviral cytokine production. Specifically, &#947;HV68 infection activates IKK&#946; and that activated IKK&#946; phosphorylates RelA to accelerate RelA degradation. As such, &#947;HV68 efficiently uncouples NF&#922;B activation from its upstream activated IKK&#946;, negating antiviral cytokine gene expression. This study elucidates an intricate strategy whereby the upstream innate immune activation is intercepted by a viral pathogen to nullify the immediate downstream transcriptional activation and evade antiviral cytokine production.

We describe a protocol to measure the antiviral cytokine production in mice infected with a model herpesvirus, murine gamma herpesvirus 68 (&#947;HV68) that is closely-related to human Kaposi&#8217;s sarcoma-associated herpesvirus (KSHV) and Epstein-Barr virus (EBV). Utilizing genetically modified mouse strains and mouse embryonic fibroblasts (MEFs), we assessed the antiviral cytokine production both in vivo and ex vivo. &#8220;Reconstituting&#8221; the expression of innate immune components in knockout embryonic fibroblasts by lentiviral transduction, we further pinpoint specific innate immune molecules and dissect the key signaling events that differentially regulate the antiviral cytokine production.

Dissection Innate Immune signalisation Evasion virale de la production de cytokines

In response to a viral infection, the host innate immune response is activated to up-regulate gene expression and production of antiviral cytokines. ...

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

An attenuated West Nile virus (WNV), a nonstructural (NS) 4B-P38G mutant, induced higher innate cytokine and T cell responses than the wild-type WNV in mice. Recently, myeloid differentiation factor 88 (MyD88) signaling was shown to be important for initial T cell priming and memory T cell development during WNV NS4B-P38G mutant infection. In this study, two flow cytometry-based methods &#8211; an in vitro T cell priming assay and an intracellular cytokine staining (ICS) &#8211; were utilized to assess dendritic cells (DCs) and T cell functions. In the T cell priming assay, cell proliferation was analyzed by flow cytometry following co-culture of DCs from both groups of mice with carboxyfluorescein succinimidyl ester (CFSE) - labeled CD4+ T cells of OTII transgenic mice. This approach provided an accurate determination of the percentage of proliferating CD4+ T cells with significantly improved overall sensitivity than the traditional assays with radioactive reagents. A microcentrifuge tube system was used in both cell culture and cytokine staining procedures of the ICS protocol. Compared to the traditional tissue culture plate-based system, this modified procedure was easier to perform at biosafety level (BL) 3 facilities. Moreover, WNV- infected cells were treated with paraformaldehyde in both assays, which enabled further analysis outside BL3 facilities. Overall, these in vitro immunological assays can be used to efficiently assess cell-mediated immune responses during WNV infection.

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

Two flow cytometry-based methods &#8211; an in vitro T cell priming assay and intracellular cytokine staining were utilized to measure antigen presenting capacity of dendritic cells and antigen-specific T cell responses to a West Nile virus mutant infection in mice.

Analyse in vitro de la réponse immunitaire cellulaire Myd88 médiation au virus du Nil occidental souche mutante Infection

An attenuated West Nile virus (WNV), a nonstructural (NS) 4B-P38G mutant, induced higher innate cytokine and T cell responses than the wild-type WNV in ...

Non-invasive Imaging of the Innate Immune Response in a Zebrafish Larval Model of Streptococcus iniae Infection

The aquatic pathogen, Streptococcus iniae, is responsible for over 100 million dollars in annual losses for the aquaculture industry and is capable of causing systemic disease in both fish and humans. A better understanding of S. iniae disease pathogenesis requires an appropriate model system. The genetic tractability and the optical transparency of the early developmental stages of zebrafish allow for the generation and non-invasive imaging of transgenic lines with fluorescently tagged immune cells. The adaptive immune system is not fully functional until several weeks post fertilization, but zebrafish larvae have a conserved vertebrate innate immune system with both neutrophils and macrophages. Thus, the generation of a larval infection model allows the study of the specific contribution of innate immunity in controlling S. iniae infection.
The site of microinjection will determine whether an infection is systemic or initially localized. Here, we present our protocols for otic vesicle injection of zebrafish aged 2-3 days post fertilization as well as our techniques for fluorescent confocal imaging of infection. A localized infection site allows observation of initial microbe invasion, recruitment of host cells and dissemination of infection. Our findings using the zebrafish larval model of S. iniae infection indicate that zebrafish can be used to examine the differing contributions of host neutrophils and macrophages in localized bacterial infections. In addition, we describe how photolabeling of immune cells can be used to track individual host cell fate during the course of infection.

Non-invasive Imaging of the Innate Immune Response in a Zebrafish Larval Model of Streptococcus iniae Infection

Here, we present a protocol for the generation and imaging of a localized bacterial infection in the zebrafish otic vesicle.

Imagerie non invasive de la réponse immunitaire innée dans un modèle de poisson zèbre larves de<em&gt; Streptococcus iniae</em&gt; Infection

The aquatic pathogen, Streptococcus iniae, is responsible for over 100 million dollars in annual losses for the aquaculture industry and is capable of ...

Using Phylogenetic Analysis to Investigate Eukaryotic Gene Origin

Phylogenetic analysis uses nucleotide or amino acid sequences or other parameters, such as domain sequences and three-dimensional structure, to construct a tree to show the evolutionary relationship among different taxa (classification units) at the molecular level. Phylogenetic analysis can also be used to investigate domain relationships within an individual taxon, particularly for organisms that have undergone substantial change in morphology and physiology, but for which researchers lack fossil evidence due to the organisms&#39; long evolutionary history or scarcity of fossilization.
In this text, a detailed protocol is described for using the phylogenetic method, including amino acid sequence alignment using Clustal Omega, and subsequent phylogenetic tree construction using both Maximum Likelihood (ML) of Molecular Evolutionary Genetics Analysis (MEGA) and Bayesian Inference via MrBayes. To investigate the origin of eukaryotic Sugars Will Eventually be Exported Transporters (SWEET) genes, 228 SWEETs including 35 SWEET proteins from unicellular eukaryotes and 57 SemiSWEET proteins from prokaryotes were analyzed. Interestingly, SemiSWEETs were found in prokaryotes, but SWEETs were found in eukaryotes. Two phylogenetic trees constructed using theoretically distinct methods have consistently&#160;suggested that the first eukaryotic SWEET gene might stem from the fusion of a bacterial SemiSWEET gene and an archaeal SemiSWEET gene.&#160;It is worth noting that one should be cautious to draw a conclusion based only on phylogenetic analysis, although it is useful to explain the underlying relationship between different taxa, which is difficult or even impossible to discern through experimental means.

A method of constructing a phylogenetic tree based on sequence homology of SWEETs from eukaryotes and SemiSWEETs from prokaryotes is described. Phylogenetic analysis is a useful tool for explaining the evolutionary relatedness between homologous proteins or genes from different organism groups.

À l’aide de l’analyse phylogénétique pour étudier l’origine de gènes eucaryotes

Phylogenetic analysis uses nucleotide or amino acid sequences or other parameters, such as domain sequences and three-dimensional structure, to construct ...

Two-photon Intravital Imaging of Leukocytes During the Immune Response in Lipopolysaccharide-treated Mouse Liver

Sepsis is a type of severe infection that can cause organ failure and tissue damage. Although the mortality and morbidity rates associated with sepsis are extremely high, no direct treatment or organ-related mechanism has been examined in detail in real time. The liver is the key organ that manages toxins and infections in the human body. Herein, we aimed to perform intravital imaging of mouse liver after induction of endotoxemia in order to track the motility of immune cells, such as neutrophils and liver capsular macrophages (LCMs). Accordingly, we designed a novel surgical method for exposure of the liver with minimally invasive surgery. Mice were intraperitoneally injected with lipopolysaccharide (LPS), a common endotoxin. Using our novel surgical approach for exposure and intravital imaging of the mouse liver, we found that neutrophil recruitment in LPS-treated LysM-green fluorescent protein (GFP) mouse liver was increased compared with that in phosphate-buffered saline-treated liver. After LPS treatment, the number of neutrophils increased significantly with time. Additionally, using CX3Cr1-GFP mice, we successfully visualized liver resident macrophages called LCMs. Therefore, to investigate the efficacy of new reagents to control immune mobility in vivo, determining the motility and morphology of neutrophils and LCMs in the liver may allow us to identify therapeutic effect in organ failure and tissue damage caused by leukocytes activation in sepsis.

We established a novel surgical protocol for two-photon imaging of live mice liver with minimal invasion. With this technique, we identified the detailed structure of the liver during lipopolysaccharide-induced endotoxemia. We anticipate that this method may be utilized to determine the effectiveness of various reagents treatment to hepatic leukocyte migration.

Two-photon Imaging intravitale des leucocytes au cours de la réponse immunitaire dans le foie de souris traitées Lipopolysaccharide

Sepsis is a type of severe infection that can cause organ failure and tissue damage. Although the mortality and morbidity rates associated with sepsis are ...