प्रतिरक्षा प्रतिक्रिया और नींद में मात्रात्मक मापन<em> ड्रोसोफिला</em
Summary
प्रतिरक्षा प्रतिक्रिया और व्यवहार के बीच की कड़ी को समझने के लिए, हम में हरकत व्यवहार को मापने के लिए एक विधि का वर्णन<em> ड्रोसोफिला</em> के रूप में के रूप में अच्छी तरह से मक्खियों की क्षमता के लिए निगरानी अस्तित्व, बैक्टीरियल लोड, और वास्तविक समय सहज रोगक्षमता, NFκB के एक महत्वपूर्ण नियामक की गतिविधि के द्वारा एक प्रतिरक्षा प्रतिक्रिया माउंट बैक्टीरियल संक्रमण के दौरान.
Abstract
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 NFκB, 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 NFκB 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.
Protocol
Representative Results
Discussion
इस प्रोटोकॉल की जांच कैसे व्यवहार विशेष रूप से सो, प्रतिरक्षा प्रतिक्रिया मापदंडों से जुड़ा हुआ है के लिए एक दृष्टिकोण की रूपरेखा. इन मानकों जीवाणु लोड, अस्तित्व परिणाम, और NFκB गतिविधि vivo में luciferase रिप?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
अनुदान IOS-1025627 # के तहत यह काम राष्ट्रीय विज्ञान फाउंडेशन द्वारा समर्थित किया गया और अनुदान के तहत स्वास्थ्य के राष्ट्रीय संस्थानों द्वारा # 1R21NS078582-01 जबड़े
Materials
| Material Name | Company | Catalogue number | Comments |
| Equipment | |||
| Incubators | Percival Scientific, Inc. | I30BLLC8 I36VLC8 |
Any incubator capable of running programmed light/temperature schedules is appropriate. |
| Drosophila Activitiy Monitors | Trikinetics Inc., Waltham, MA | DAM2 | As described elsewhere6, this system requires a computer interface, software, and other accessories. |
| Pyrex Glass Tubes | Trikinetics Inc., Waltham, MA | PGT-5×65 | |
| Microplate scintillation and luminescence counter | Perkin Elmer | TopCount NXT 12 detector |
Any microplate reader capable of detecting luminescence can be used for this type of reporter assay. TopCount contains multiple detectors and an automated stacker; it is capable of being programmed to read continuously from multiple plates. |
| FluorChem 8900 | Alpha Innotech | Imaging of bacterial cultures is optional; any digital imaging system with visual light capability is sufficient. | |
| Micropipette Puller | Tritech Research, Inc. | Narishige PC-10 | |
| Supplies | |||
| Borosilicate Glass Capillaries | World Precision Instrument Inc. | 1B100F-4 | |
| 3 ml Syringe | Fisher Scientific | BD 305482 | |
| Syringe Needles | Fisher Scientific | BD 305196 | 18 G – cut off the tip of the needle to prevent damage to the tubing. |
| Silicone Tubing, i.d. (0.030″) o.d. (0.065″) Wall Thickness (0.018″) | VWR | 60985-706 | Used for attaching glass capillary needles to a syringe |
| 3 Way Stopcock | American Pharmaseal Company | K75 | |
| Kontes Pellet Pestle Cordless Motor | Fisher Scientific | K749540-0000 | |
| Kontes Pellet Pestle | Fisher Scientific | K749521-1590 | |
| Glass balls 3mm | VWR | 26396-630 | |
| Microplate Microlite 1+ | Thermo Scientific | 7571 | Select 96-well plates that are appropriate for luminescence – they must be opaque. |
| TopSeal-A:96-well Microplates | PerkinElmer | 6005185 | Microplate Press-On Adhesive Sealing Film |
| D-Luciferin, Potassium Salt | Gold BioTechnology, Inc. | LUCNA | |
| Software | |||
| Insomniac2 | Available upon request to the authors | custom; written by Lesley Ashmore, Ph.D. (Westminster College) | Matlab based software that has been used routinely for analysis of sleep2,6,11 |
| Drosonex | Available upon request to the authors | custom; written by Thomas Coradetti (Sidewalk Software) | A PC MSVC6 program used for survival analysis from raw data files collected with the Trikinetics system |
| Photoshop CS3 | Adobe | Useful for obtaining numbers of cfu/plate from digital images (optional) |
References
- Majde, J. A., Krueger, J. M. Links between the innate immune system and sleep. J. Allergy Clin. Immunol. 116, 1188-1198 (2005).
- Kuo, T. H., Pike, D. H., Beizaeipour, Z., Williams, J. A. Sleep triggered by an immune response in Drosophila is regulated by the circadian clock and requires the NFkappaB Relish. BMC Neurosci. 11, 1471-2202 (2010).
- Preston, B. T., Capellini, I., McNamara, P., Barton, R. A., Nunn, C. L. Parasite resistance and the adaptive significance of sleep. BMC Evol Biol. 9, 7 (2009).
- Imeri, L., Opp, M. R. How (and why) the immune system makes us sleep. Nat. Rev. Neurosci. 10, 199-210 (2009).
- Ayres, J. S., Schneider, D. S. A Signaling Protease Required for Melanization in Drosophila Affects Resistance and Tolerance of Infections. PLoS Biol. 6, e305 (2008).
- Chiu, J. C., Low, K. H., Pike, D. H., Yildirim, E., Edery, I. Assaying Locomotor Activity to Study Circadian Rhythms and Sleep Parameters in Drosophila. J. Vis. Exp. (43), e2157 (2010).
- Lee, J. E., Edery, I. Circadian Regulation in the Ability of Drosophila to Combat Pathogenic Infections. Curr. Biol. 18, 195-199 (2008).
- Stone, E. F., et al. The circadian clock protein timeless regulates phagocytosis of bacteria in Drosophila. PLoS Pathog. 8, e1002445 (2012).
- Hill-Burns, E. M., Clark, A. G. X-linked variation in immune response in Drosophila melanogaster. Génétique. 183, 1477-1491 (2009).
- Short, S. M., Lazzaro, B. P. Female and male genetic contributions to post-mating immune defence in female Drosophila melanogaster. Proc. Biol. Sci. 277, 3649-3657 (2010).
- Williams, J. A., Sathyanarayanan, S., Hendricks, J. C., Sehgal, A. Interaction between sleep and the immune response in Drosophila: a role for the NFkappaB relish. Sleep. 30, 389-400 (2007).
- Ramsden, S., Cheung, Y. Y., Seroude, L. Functional analysis of the Drosophila immune response during aging. Aging Cell. 7, 225-236 (2008).
- Williams, J. A., Su, H. S., Bernards, A., Field, J., Sehgal, A. A circadian output in Drosophila mediated by neurofibromatosis-1 and Ras/MAPK. Science. 293, 2251-2256 (2001).
- Hedengren, M., et al. Relish, a central factor in the control of humoral but not cellular immunity in Drosophila. Mol. Cell. 4, 827-837 (1999).
- Leulier, F., Rodriguez, A., Khush, R. S., Abrams, J. M., Lemaitre, B. The Drosophila caspase Dredd is required to resist gram-negative bacterial infection. EMBO Rep. 1, 353-358 (2000).
- Wu, L. P., Choe, K. -. M., Lu, Y., Anderson, K. V. Drosophila Immunity: Genes on the Third Chromosome Required for the Response to Bacterial Infection. Génétique. 159, 189-199 (2001).
- Dionne, M. S., Ghori, N., Schneider, D. S. Drosophila melanogaster is a genetically tractable model host for Mycobacterium marinum. Infect. Immun. 71, 3540-3550 (2003).
- Romeo, Y., Lemaitre, B. Drosophila immunity: methods for monitoring the activity of Toll and Imd signaling pathways. Methods Mol. Biol. 415, 379-394 (2008).
- Lu, Y., Wu, L. P., Anderson, K. V. The antibacterial arm of the Drosophila innate immune response requires an I{kappa}B kinase. Genes Dev. 15, 104-110 (2001).
- Koh, K., Evans, J. M., Hendricks, J. C., Sehgal, A. A Drosophila model for age-associated changes in sleep:wake cycles. Proc. Natl. Acad. Sci. U. S. A. 103, 13843-13847 (2006).
- Bushey, D., Hughes, K. A., Tononi, G., Cirelli, C. Sleep, aging, and lifespan in Drosophila. BMC Neurosci. 11, 1471-2202 (2010).
- Allee, W. C. . The social life of animals. , (1938).
- Stanewsky, R., Jamison, C. F., Plautz, J. D., Kay, S. A., Hall, J. C. Multiple circadian-regulated elements contribute to cycling period gene expression in Drosophila. Embo. J. 16, 5006-5018 (1997).
- Levine, J. D., Funes, P., Dowse, H. B., Hall, J. C. Signal analysis of behavioral and molecular cycles. BMC Neurosci. 3, 1 (2002).
- Ayres, J. S., Schneider, D. S. The role of anorexia in resistance and tolerance to infections in Drosophila. PLoS Biol. 7, e1000150 (2009).
- Plautz, J. D., Kaneko, M., Hall, J. C., Kay, S. A. Independent photoreceptive circadian clocks throughout Drosophila. Science. 278, 1632-1635 (1997).
- Huber, R., et al. Sleep homeostasis in Drosophila melanogaster. Sleep. 27, 628-639 (2004).
- Zimmerman, J. E., Raizen, D. M., Maycock, M. H., Maislin, G., Pack, A. I. A video method to study Drosophila sleep. Sleep. 31, 1587-1598 (2008).
- Toth, L. A., Rehg, J. E., Webster, R. G. Strain differences in sleep and other pathophysiological sequelae of influenza virus infection in naive and immunized mice. J. Neuroimmunol. 58, 89-99 (1995).
- Hendricks, J. C., et al. Rest in Drosophila is a sleep-like state. Neuron. 25, 129-138 (2000).
- Wu, M. N., Koh, K., Yue, Z., Joiner, W. J., Sehgal, A. A genetic screen for sleep and circadian mutants reveals mechanisms underlying regulation of sleep in Drosophila. Sleep. 31, 465-472 (2008).
