Summary

Fare Kemik İliği monositlerin Takip<em> İn Vivo</em

Published: February 27, 2015
doi:

Summary

Monocytes are key regulators of innate immunity and play a critical role in the renewal of the peripheral mononuclear phagocytic system and in case of inflammation. This manuscript describes the procedure of real time imaging of the mouse calvaria bone marrow to study the monocyte mobilisation mechanism.

Abstract

Real time multiphoton imaging provides a great opportunity to study cell trafficking and cell-to-cell interactions in their physiological 3-dimensionnal environment. Biological activities of immune cells mainly rely on their motility capacities. Blood monocytes have short half-life in the bloodstream; they originate in the bone marrow and are constitutively released from it. In inflammatory condition, this process is enhanced, leading to blood monocytosis and subsequent infiltration of the peripheral inflammatory tissues. Identifying the biomechanical events controlling monocyte trafficking from the bone marrow towards the vascular network is an important step to understand monocyte physiopathological relevance. We performed in vivo time-lapse imaging by two-photon microscopy of the skull bone marrow of the Csf1r-Gal4VP16/UAS-ECFP (MacBlue) mouse. The MacBlue mouse expresses the fluorescent reporters enhanced cyan fluorescent protein (ECFP) under the control of a myeloid specific promoter 1, in combination with vascular network labelling. We describe how this approach enables the tracking of individual medullar monocytes in real time to further quantify the migratory behaviour within the bone marrow parenchyma and the vasculature, as well as cell-to-cell interactions. This approach provides novel insights into the biology of the bone marrow monocyte subsets and allows to further address how these cells can be influenced in specific pathological conditions.

Introduction

The bone marrow plays a central role in hematopoiesis and represents the main reservoir of monocytes that constitutively recirculate between the blood and the medullar parenchyma, renew the pool of circulating monocytes with a short life span 2,3 and participate in the reconstitution of the steady state tissue-macrophages and dendritic cells 4. During inflammation or after transient aplasia, monocytes are actively mobilized from either the bone marrow or the spleen 5, 6, 7 and colonize inflamed organs. Several chemoattractant axis have been involved in the process of myeloid cell mobilization from the bone marrow 8, 5, 6,9. Beyond the myeloid compartment the bone marrow is also an important site of T lymphocyte priming 10 and a niche of immunological memory 11,12. Thus, this tissue is central for numerous investigations in the field of hematology and immunology. Our knowledge on the structural organization of medullar myeloid cells mainly arises from the analysis of histological section of fixed tissues 13. This static view does not allow for a study of the cellular exchange dynamic between the different compartments of the bone marrow, which is the basis of its functional activity.

Intravital imaging constitutes an important biological input in the study of cell mobility, cell adherence and cell-to-cell interactions, which were previously described only from in vitro systems. Technical challenges for proper intravital imaging include the ability to reach the tissue of interest in an optical point of view, and to maximize its isolation from physiological (breath, muscle or peristaltic contractions) or mechanical drifts (tissue disruption and extension following surgery, and exposure to microscope objective as well as temperature and vascular/oxygenation perturbations). Microscopic drifts may limit the ability to keep the focus a long time and could introduce artifacts in the quantification of cell motility. One alternative, validated for several tissues to reduce these technical difficulties, is to work on explanted tissue incubated in a thermostated and oxygenated medium; however, complete disruption of the lymphatic and vascular circulation may be problematic. Intravital imaging of skull bone marrow has several advantages concerning these issues. Firstly, it requires minimal surgical action. Secondly, thickness of the bone in this region allows direct visualization of bone marrow niches without abrasion, thus reducing physiological perturbations. The medullar network can be imaged in the parasagittal region of the bone; however the sinusoids are more visible in the fronto-parietal area where the bone matrix is thinner12,14.

Intravital imaging relies on the availability of the most accurate fluorescent reporter tagging the population of interest. In vitro labelling of purified cell population before adoptive transfer led to important characterization of hematopoietic stem cell niches 15 or bone marrow endothelial microdomains favouring tumor engraftment 16, and provided several fundamental inputs on key concepts in immunology 17 . However, this approach usually requires hundreds of thousands of cells to get a chance to detect them afterwards in vivo. This could be explained by the high mortality rate following staining, the dilution in the whole body and the change in the activation state, which might lead to biased homing. Endogenous tagging from transgenic mouse system greatly overcomes these limitations and has allowed to image the behaviour of endogenous osteoblast 8, megakaryocytes 18 or myeloid-lineage subsets 6 . Nevertheless, one has to be cautious when considering the specificity of the fluorescent reporter among the studied subset.

The Csf1r-Gal4VP16/UAS-ECFP, called MacBlue mouse 1, is a valuable transgenic system to study medullar monocytes with real time imaging 6. Intravenous injection of high molecular weight rhodamin-dextran distinguishes the medullar parenchyma from the vascular sinusoid network of the bone marrow. Using this approach, it is possible to track the monocyte behaviour in the different medullar compartments in a specific physiopathological context of interest. Furthermore, we propose an additional strategy to compare monocyte dynamics with that of neutrophils through in vivo labelling using a specific antibody.

Protocol

NOT: Tüm deney protokolleri Fransız Hayvan Deneyleri Etik Kurul tarafından onaylanan ve sayı A-75-2065 ile "Hizmet Koruma et Santé Animales, Çevre'den" tarafından doğrulanmıştır. Örnek boyutları deneylerin tekrarlanabilirliğini sağlamak için seçilmiş ve hayvan etiği düzenleme 3R göredir. Mouse 1. Hazırlık Anestezi Görüntüleme kısa bir süre (en az 1 saat) için, ketamin (100 mg / kg) ve ketamin (10 mg / kg) ihtiva eden bir t…

Representative Results

Fare kafatası yapısı intravital görüntüleme, kemik iliği fizyoloji çalışmak için iyi bir fırsat sunuyor. Ön-parietal alanı çevresinde ince olan kemik, kemiğin aşınma olmadan medulla nişler erişim elde etmek mümkündür. 1 MacBlue transgenik fare kafatası geniş bir 2D alanını temsil Şekil. kemik matriksi esas olarak SHG 19 tarafından kolayca tespit edilebilir kollajen oluşmaktadır. Rodaminle Dekstranın Enjeksiyon kemik iliği damar ağı lekeleri …

Discussion

in vivo görüntüleme yönteminin kritik noktaları görüntüleme süresini maksimize etmek ve inflamatuar hücrelerin dinamiklerini etkileyebilecek bakteriyel kontaminasyon ve inflamasyon riskini en aza indirmek için odak istikrarı sağlamak için vardır. Cerrahi kemik iliği erişmek için minimal gerçekleştirilen gibi kafatası kemik iliği Görüntüleme bu amaçlarını izler. Steril malzeme ve antiseptik kullanımı hücre homeostazındaki pertürbasyon neden olabilecek enfeksiyon riskini s?…

Disclosures

The authors have nothing to disclose.

Acknowledgements

Yazarlar editoryal yardım için Anne Daron ve Pierre Louis Loyher teşekkür etmek istiyorum, iki-foton mikroskop ve yardım damızlık fareler için hayvan Facility "NAC" ve Camille Baudesson yardım için Plateforme Imagerie Pitié-Salpêtrière (PICPS). Hibe sözleşmesi kapsamında bu sonuçlara önde gelen araştırma Avrupa Topluluğu'nun Yedinci Çerçeve Programı fon aldı (FP7 / 2007-2013) n 304.810 ° – baskınlar, ve n Université Pierre et Marie Curie gelen Inserm den 241.440-Endostem, ° "Ortaya "la gelen" "dan," Ligue contre le kanser Derneği la Recherche sur le Kanser dökün "ve gelen" Agence Nationale de la Recherche "Program Doğuşu 2012 (ANR-EMMA-050). PH la "Ligue contre le kanser" tarafından desteklendi.

Materials

Name of Material/ Equipment Company Catalog Number Comments/Description
Ketamin Merial 100mg/mL, anesthetic
Xylazin Bayer HealthCare 10mg/mL, anesthetic
Isofluran Baxter 2.5%, anesthetic
O2/NO2 70/30 mixture, anesthetic
Rhodamin-Dextran Invitrogen 2MDa, 10mg/mL, Vascular staining
Ly6G-PE Becton-Dickinson clone 1A8, neutrophils staining
Stereotactic holder Home made surgery
Ethanol 70% surgery
Sterile scissors and nippers surgery
Rubber ring 18mm diameter, surgery
Glubran 2 Queryo Medical Surgical Glue, rubber ring fixation
Small gauge needles Terumo surgery
Zeiss LSM 710 NLO multiphoton microscope  Carl Zeiss Microscope
Ti:Sapphire crystal laser  Coherent Chameleon Ultra 140fs pulses of NIR light
Zen 2010 Carl Zeiss Acquistion Software
Imaris Bitplane  Bitplane Analysis Software, 3D automatic tracking
PBS 1X D. Dutscher surgery
Thermostated chamber Carl Zeiss intravital imaging

References

  1. Ovchinnikov, D. A., et al. Expression of Gal4-dependent transgenes in cells of the mononuclear phagocyte system labeled with enhanced cyan fluorescent protein using Csf1r-Gal4VP16/UAS-ECFP double-transgenic mice. J Leukoc Biol. 83, 430-433 (2008).
  2. Parihar, A., Eubank, T. D., Doseff, A. I. Monocytes and macrophages regulate immunity through dynamic networks of survival and cell death. J Innate Immun. 2, 204-215 (2010).
  3. Geissmann, F., Jung, S., Littman, D. R. Blood monocytes consist of two principal subsets with distinct migratory properties. Immunity. 19, 71-82 (2003).
  4. Robbins, C. S., Swirski, F. K. The multiple roles of monocyte subsets in steady state and inflammation. Cell Mol Life Sci. 67, 2685-2693 (2010).
  5. Shi, C., et al. Bone marrow mesenchymal stem and progenitor cells induce monocyte emigration in response to circulating toll-like receptor ligands. Immunity. 34, 590-601 (2011).
  6. Jacquelin, S., et al. CX3CR1 reduces Ly6Chigh-monocyte motility within and release from the bone marrow after chemotherapy in mice. Blood. 122, 674-683 (2013).
  7. Swirski, F. K., et al. Identification of splenic reservoir monocytes and their deployment to inflammatory sites. Science. 325, 612-616 (2009).
  8. Germain, R. N., Miller, M. J., Dustin, M. L., Nussenzweig, M. C. Dynamic imaging of the immune system: progress, pitfalls and promise. Nat Rev Immunol. 6, 497-507 (2006).
  9. Charo, I. F., Ransohoff, R. M. The many roles of chemokines and chemokine receptors in inflammation. N Engl J Med. 354, 610-621 (2006).
  10. Milo, I., et al. Dynamic imaging reveals promiscuous crosspresentation of blood-borne antigens to naive CD8+ T cells in the bone marrow. 122, 193-208 (2013).
  11. Cavanagh, L. L., et al. Activation of bone marrow-resident memory T cells by circulating, antigen-bearing dendritic cells. Nat Immunol. 6, 1029-1037 (2005).
  12. Mazo, I. B., et al. Bone marrow is a major reservoir and site of recruitment for central memory CD8+ T cells. Immunity. 22, 259-270 (2005).
  13. Travlos, G. S. Normal structure, function, and histology of the bone marrow. Toxicol Pathol. 34, 548-565 (2006).
  14. Mazo, I. B., et al. Hematopoietic progenitor cell rolling in bone marrow microvessels: parallel contributions by endothelial selectins and vascular cell adhesion molecule 1. J Exp Med. 188, 465-474 (1998).
  15. Rashidi, N. M., et al. In vivo time-lapse imaging of mouse bone marrow reveals differential niche engagement by quiescent and naturally activated hematopoietic stem cells. Blood. , (2014).
  16. Sipkins, D. A., et al. In vivo imaging of specialized bone marrow endothelial microdomains for tumour engraftment. Nature. 435, 969-973 (2005).
  17. Cariappa, A., et al. Perisinusoidal B cells in the bone marrow participate in T-independent responses to blood-borne microbes. Immunity. 23, 397-407 (2005).
  18. Junt, T., et al. Dynamic visualization of thrombopoiesis within bone marrow. Science. 317, 1767-1770 (2007).
  19. Zoumi, A., Yeh, A., Tromberg, B. J. Imaging cells and extracellular matrix in vivo by using second-harmonic generation and two-photon excited fluorescence. Proc Natl Acad Sci U S A. 99, 11014-11019 (2002).
  20. Miller, M. J., Wei, S. H., Parker, I., Cahalan, M. D. Two-photon imaging of lymphocyte motility and antigen response in intact lymph node. Science. 296, 1869-1873 (2002).
  21. Cahalan, M. D., Parker, I. Choreography of cell motility and interaction dynamics imaged by two-photon microscopy in lymphoid organs. Annu Rev Immunol. 26, 585-626 (2008).
  22. Yost, C. C., et al. Impaired neutrophil extracellular trap (NET) formation: a novel innate immune deficiency of human neonates. Blood. 113, 6419-6427 (2009).
  23. Devi, S., et al. Multiphoton imaging reveals a new leukocyte recruitment paradigm in the glomerulus. Nat Med. 19, 107-112 (2013).

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Cite This Article
Hamon, P., Rodero, M. P., Combadière, C., Boissonnas, A. Tracking Mouse Bone Marrow Monocytes In Vivo. J. Vis. Exp. (96), e52476, doi:10.3791/52476 (2015).

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