Immunofluorescence de suivi de l'absorption cellulaire de la lactoferrine humaine et de son activité associée antivirale contre le virus de l'hépatite C
Summary
The human lactoferrin (hLF) is a component of the immune system. In this study, immunofluorescence assays are used to demonstrate both the hepatocellular uptake of hLF and a qualitative reduction in the hepatitis C virus replication upon treatment with hLF.
Abstract
L'immunofluorescence est une technique de laboratoire couramment utilisés pour étudier de nombreux aspects de la biologie. Il est généralement utilisé pour visualiser la distribution et / ou la localisation d'une molécule cible dans des cellules et des tissus. Immunofluorescence repose sur la spécificité des anticorps marqués par fluorescence contre leurs antigènes correspondants au sein d'une cellule. Les deux approches directes et indirectes immunofluorescence peuvent être utilisés qui reposent sur l'utilisation d'anticorps liés à un fluorochrome. L'immunofluorescence directe est moins fréquemment utilisé, car il fournit le signal inférieur, implique un coût plus élevé et moins de flexibilité. En revanche, l'immunofluorescence indirecte est plus couramment utilisé en raison de sa haute sensibilité et fournit un signal amplifié depuis plus d'un anticorps secondaire peut se rattacher à chaque anticorps primaire. Dans ce manuscrit, à la fois microscopie à épifluorescence et microscopie confocale ont été utilisés pour contrôler l'internalisation de la lactoferrine humaine, un élément important de la réponse immunitairesystème, dans les cellules hépatiques. De plus, nous avons surveillé le potentiel d'inhibition de la hLF sur la réplication intracellulaire du virus de l'hépatite C par immunofluorescence. Aussi bien les avantages et les inconvénients associés à ces approches sont discutées.
Introduction
Immunofluorescence is a technique that uses a fluorescence microscope to visualize the distribution and/or localization of a target molecule in a biological sample. Immunofluorescence relies on the specificity of fluorescent-labelled antibodies against their corresponding antigens within a cell1. It is typically used on tissue sections and cultured cell lines in order to analyze the distribution/localization of various biological molecules such as proteins, nucleic acids and glycans. It should be noted that immunofluorescence is often used in combination with other non-antibody methods of fluorescent staining such as the 4′,6-diamidino-2-phenylindole (DAPI) stains which are typically used to label DNA2. Moreover, this technique involves fixation of the cells which allows the analysis of cells at a specific time.
Different types of microscopes can be used to analyze immunofluorescence samples. The simplest is the epifluorescence microscope (Figure 1) for which excitation of the fluorochrome and detection of the fluorescence are done through the same light path3. Because most of the excitation light is transmitted through the sample, only reflected excitatory light can reach the objective together with the emitted light. This approach unfortunately leadsto a frequent high signal to noise ratio.In contrast, confocal microscopy (Figure 2) offers a distinct advantage for increasing optical resolution and contrast by means of adding a spatial pinhole placed at the confocal plane of the lens to eliminate out-of-focus light4. This approach allows the reconstruction of three-dimensional structures from the obtained images. However, since an important fraction of the light from the sample is blocked at the pinhole, long exposures are often required.
There are two classes of immunofluorescence techniques, primary (or direct) and secondary (or indirect). Direct immunofluorescence involves a primary antibody linked with a fluorochrome (Figure 3). This method is less frequently used because it provides lower signal, involves higher cost and less flexibility1. Moreover, such antibodies are generally harder to find commercially. On the other hand, the direct attachment of the fluorochrome to the antibody significantly reduces the number of steps in the procedure, saving time and frequently reducing non-specific background signal. This also limits the possibility of antibody cross-reactivty.
Indirect immunofluorescence involves a primary unlabelled antibody which is specific for the epitope of interest1. A secondary antibody which carries the fluorochrome then recognizes the primary antibody and binds to it (Figure 3). Although indirect immunofluorescence is more complex and time consuming than direct immunofluorescence, it is frequently used because of its high sensitivity and it also provides an amplified signal since more than one secondary antibody can attach to each primary antibody. In addition, a vast array of commercial secondary antibodies is available at affordable prices.
Hepatitis C virus (HCV) is a major public-health problem with 130-170 million individuals chronically infected worldwide. In order to halt the epidemic, therapy against HCV will need to be both effective and widely available. Studies focusing on safe and affordable natural product active against HCV have revealed the antiviral activity of the human Lactoferrin (hLF) protein which binds and neutralizes the circulating virion5. In the current study, investigation of hLF activity on the HCV subgenomic replicon system, which is independent from viral entry and shedding, revealed a distinct antireplicative activity of hLF against HCV. This manuscript presents a study in which immunofluorescence assays were performed to monitor the internalization of hLF, an important component of the immune system6, into hepatic cells. Moreover, we monitored the inhibitory potential of hLF on the intracellular replication of the Hepatitis C virus (HCV).
Protocol
Representative Results
Discussion
L'épidémie de VHC demeure une menace mondiale, avec 80% des patients nouvellement infectés en développement une infection chronique, les mettant à risque de cirrhose, l'insuffisance hépatique ou carcinome hépatocellulaire. Action directe des agents antiviraux ciblant la réplication du VHC et de la maturation représentent premiers agents anti-VHC, comme l'a récemment démontré par l'approbation réglementaire de deux inhibiteurs de la protéase NS3 extrémité N-terminale (bocéprévir et téla…
Disclosures
The authors have nothing to disclose.
Acknowledgements
This work was funded by both the Canadian Institutes of Health Research and Natural Sciences and the Engineering Research Council of Canada. M. Bisaillon is a Chercheur Boursier Senior from the Fonds de Recherche en Santé du Québec and also a member of the Centre de Recherche Clinique du Centre Hospitalier Universitaire de Sherbrooke. We thank Dr. Ralf Bartenschlager for the generous gift of the HCV replicon system. We also thank Dr. Charles Rice and Dr. Daniel Lamarre for kindly providing the hepatic cell line. We also want to thank Guillaume Tremblay for technical assistance.
Materials
| DMEM | Wisent | 319-005-CL | |
| PAF | BioShop | PAR070.1 | Flammable solid, skin irritant, lungs and eyes. |
| PBS | Wisent | 311-425-CL | Without Ca2+ & Mg2+ |
| NGS | Wisent | 053-150 | |
| AlexaFluor 488-labeled anti-mouse | Invitrogen | A11017 | |
| AlexaFluor 568-labeled anti-rabbit | Invitrogeb | A21069 | |
| Wheat germ agglutinin Alexa Fluor 488 conjugate (WGA) | Invitrogen | W11261 | Potentially mutagenic |
| Anti-NS5A rabbit | Abcam | ab2594 | |
| Anti-hLF mouse | Abcam | ab10110 | |
| SlowFade | Invitrogen | S36937 | |
| Hoechst stain | Life Techn. | H1399 | Potentially mutagenic and carcinogenic |
| hLF | Sigma | L0520 | |
| Nikon Eclipse visible/epifluorescence Microscope | Nikon | TE2000-E | |
| epifluorescence/confocal microscope | Olympus | FV1000 |
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