Depletion and Reconstitution of Macrophages in Mice

Summary

Macrophages play a central role in homeostasis and pathology in many tissues. The protocol presented here describes methods for depleting macrophages in vivo, deriving polarized macrophages from bone marrow aspirates, and adoptively transferring macrophages into mice. These techniques allow determination of the role that polarized macrophages play in health and disease.

Abstract

Macrophages are critical players in the innate immune response to infectious challenge or injury, initiating the innate immune response and directing the acquired immune response. Macrophage dysfunction can lead to an inability to mount an appropriate immune response and as such, has been implicated in many disease processes, including inflammatory bowel diseases. Macrophages display polarized phenotypes that are broadly divided into two categories. Classically activated macrophages, activated by stimulation with IFNγ or LPS, play an essential role in response to bacterial challenge whereas alternatively activated macrophages, activated by IL-4 or IL-13, participate in debris scavenging and tissue remodeling and have been implicated in the resolution phase of inflammation. During an inflammatory response in vivo, macrophages are found amid a complex mixture of infiltrating immune cells and may participate by exacerbating or resolving inflammation. To define the role of macrophages in situ in a whole animal model, it is necessary to examine the effect of depleting macrophages from the complex environment. To ask questions about the role of macrophage phenotype in situ, phenotypically defined polarized macrophages can be derived ex vivo, from bone marrow aspirates and added back to mice, with or without prior depletion of macrophages. In the protocol presented here clodronate-containing liposomes, versus PBS injected controls, were used to deplete colonic macrophages during dextran sodium sulfate (DSS)-induced colitis in mice. In addition, polarized macrophages were derived ex vivo and transferred to mice by intravenous injection. A caveat to this approach is that clodronate-containing liposomes deplete all professional phagocytes, including both dendritic cells and macrophages so to ensure the effect observed by depletion is macrophage-specific, reconstitution of phenotype by adoptive transfer of macrophages is necessary. Systemic macrophage depletion in mice can also be achieved by backcrossing mice onto a CD11b-DTR background, which is an excellent complementary approach. The advantage of clodronate-containing liposome-mediated depletion is that it does not require the time and expense involved in backcrossing mice and it can be used in mice regardless of the background of the mice (C57BL/6, BALB/c, or mixed background).

Protocol

1. Depleting Macrophages Using Clodronate-containing Liposomes

1. Liposomes are stored at 4 °C. Two hours prior to injection, remove clodronate-containing liposomes, PBS-containing liposomes, or sterile PBS (injection control) from the refrigerator to allow them to acclimate to room temperature (18 °C).
2. Invert the tubes containing liposomes 8-10 times to ensure an even distribution before loading 200 μl into a 1 ml syringe. Attach a 26 gauge needle to the top of the syringe.
3. Using your left hand, scruff the mouse just below the ears holding enough skin to immobilize its head and limbs.
4. Tilt the mouse so its head is....

Discussion

Macrophages are phagocytic cells that play an important role in the immune system. They are responsible for initiating the innate immune response and directing the acquired immune response. Typically, classically activated macrophages are activated by IFNγ or LPS and are responsible for eliminating pathogens and mounting an inflammatory response¹. Conversely, alternatively activated macrophages are activated by IL-4 or IL-13 and play a role in debris scavenging and tissue remodeling during the.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Clodronate-containing liposomes	Clodronateliposomes.org
PBS-containing liposomes	Clodronateliposomes.org
Sterile PBS pH7.4	Invitrogen	14190
IMDM	Invitrogen	12440
Fetal Calf Serum (FCS)	Invitrogen	12483
acetic acid	BDH Aristar	BDH3092-500MLP
Monothioglycerol (MTG)	Sigma	96-275
Recombinant murine MCSF	Stemcell Technologies	02951
Recombinant murine GM-CSF	Stemcell Technologies	02935
Recombinant murine IL-3	Stemcell Technologies	02903
Recombinant murine IFNγ	Stemcell Technologies	02746
Recombinant murine IL-4	Stemcell Technologies	02714
Cell Dissociation Buffer	Invitrogen	13150-016
Rat anti-F4/80 Antibody	AbD Serotec	MCA497GA
Mouse anti-ArgI Antibody	BD Transduction Laboratories	610708
Table 1. Specific reagents used in this protocol.