Polarization and Characterization of M1 and M2 Human Monocyte-Derived Macrophages on Implant Surfaces

Summary

Here, we present a detailed protocol for assessing the immunomodulatory potential of implant surfaces in vitro, aiming to improve the reliability and reproducibility of current protocols and promoting further research. Secretory cytokine profiles, mRNA expression, and cell surface markers were monitored using blood monocyte-derived macrophages to investigate macrophage polarization cultivated on titanium.

Abstract

Foreign body reaction (FBR), an immune-mediated complex healing process, plays a crucial role in integrating implants into the body. Macrophages, as the first line of immune system interaction with implant surfaces, play a bidirectional role in modulating the inflammation-regeneration balance. For a deep understanding and the evaluation of the reactions between implant materials and immune responses, reliable in vitro methods and protocols are pivotal. Among different in vitro models, primary monocyte-derived macrophages (MDMs) present an excellent model for investigating macrophage-implant interactions. We have implemented an experimental protocol to evaluate the polarization of MDMs into M1 (classically activated) and M2 (alternatively activated) macrophages on implant surfaces. We isolated blood monocytes from healthy donors and differentiated them into macrophages using macrophage colony-stimulating factor (M-CSF). Differentiated macrophages were cultured on implant surfaces and polarized into M1 and M2 subtypes. M1 polarization was achieved in the presence of interferon (IFN)-γ and lipopolysaccharide (LPS), while M2 polarization was performed in a medium containing interleukin (IL)-4 and IL-13. We evaluated macrophage phenotypes by Enzyme-linked Immunosorbent Assay (ELISA), confocal laser scanning microscopy (CLSM), and quantitative real-time PCR (qRT-PCR) based on panels of secreted cytokines, cell surface markers, and expressed genes. The extracted RNA was transformed into complementary DNA (cDNA), and qRT-PCR was used to quantify mRNA related to M1 and M2 macrophages. Accordingly, M1 macrophages have been characterized by higher expression of proinflammatory Tumor necrosis factor (TNF-α) cytokine and CCR7 surface marker compared to M2 macrophages, which exhibited higher levels of CD209 and CCL13. Consequently, CCR7 and CD209 were identified as specific and reliable markers of M1 and M2 macrophage subtypes by immunostaining and visualizing by CLSM. Further confirmation was achieved by ELISA detecting elevated TNF-ɑ level in M1 and increased CCL13 in M2 cells. The proposed markers and experimental setup can be used effectively to assess the immunomodulatory potential of implants.

Introduction

Implantable biomaterials have become a conventional solution for various human diseases and play a great role in biomedical research, including tissue engineering, drug delivery systems, and implants^1,2. There is a wide range of implants made from various materials with different structures and functionalities, such as hip prostheses, stents, meshes, heart valves, or dental implants. Upon implantation, the tissue-implant contact provokes an immune response, followed by resolution, tissue remodeling, and homeostasis. These processes are influenced by the physical, chemical, and bioactive characteristics of the ....

Protocol

Human peripheral blood was obtained from healthy blood donors in accordance with the protocol approved by the Ethics Committee of the medical faculty at the University of Tübingen (ethical approval: 286/2021 BO). Human PBMCs were isolated using Density Gradient Centrifugation, as previously described¹⁶. The following protocol is outlined for PBMCs isolated from 24 mL of blood. A schematic representation of the protocol is shown in Figure 1.

Discussion

A thorough understanding of macrophage behavior is essential for comprehending the immunomodulatory properties of implantable materials. Several studies have reported heterogeneous markers, a variety of cell models, and protocols for characterizing macrophage polarization in vitro^17,18,19,20. To improve reproducibility, reliability, and comparability of experimental results, standardiz.......

Materials

Name	Company	Catalog Number	Comments
24-well plate, not-treated	Corning Incorporated (Kennebunk, USA)	144530
Absorbance reader Infinite F50	TECAN Austria GmbH (Grödig, Austria)	TCAT91000001
Accutase in DPBS, 0.5 mM EDTA	EMD Millipore Corp. (Burlington, USA)	SCR005
Anti-Fade Fluorescence Mounting Medium -Aqueous, Fluoroshield	abcam (Cambridge, UK)	ab104135
Bio-Rad MJ Research PTC-200 Peltier Thermal Cycler	Bio-Rad / MJ Research	7212
Bovine serum albumin (BSA)	VWR International bvba (Leuven, Belgium)	422361V
Centrifuge 5804 R	Eppendorf SE (Hamburg, Germany)	5804 R
DC-SIGN (D7F5C) XP Rabbit mAb	Cell Signaling Technology	13193
Dimethyl sulfoxide	Sigma Aldrich Co. (St.Louis, MO, USA)	D2438-5X10ML
DRAQ5 Staining Solution	Milteny Biotec (Bergisch Gladbach, Germany)	130-117-344
Ethanol ≥99.8% for molecularbiology	Carl Roth GmbH + CO. KG (Karlsruhe, Germany)	1HPH.1
Goat Anti-Mouse IgG (H&L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488	Invitrogen	A32723TR
Goat anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, Cyanine3	Invitrogen	A10520
GraphPad Prism	GraphPad	Version  9.4.1
Human CCR7 Antibody	R&D Systems	MAB197
Human IFN-gamma Recombinant	Invitrogen (Rockford, USA)	RIFNG100
Human IL-13	Milteny Biotec (Bergisch Gladbach, Germany)	5230901032
Human IL-4	Milteny Biotec (Bergisch Gladbach, Germany)	130-095-373
Human M-CSF	Peprotech (Cranbury, USA)	300-25
Leica TCS SP5	Leica Microsystems CMS GmbH (Mannheim, Germany)	https://www.leica-microsystems.com/products/confocal-microscopes/p/leica-tcs-sp5/
Lipopolysaccharides from Escherichia	Sigma Aldrich Co. (Missouri, USA)	L4391-1MG
Luna Universal qPCR Master Mix	New England Biolabs	NEB #M3003
LunaScript RT SuperMix Kit	New England Biolabs	E3010L
Lymphocyte Separation Medium 1077	PromoCell (Heidelberg, Germany)	C-44010
MCP-4/CCL13 Human ELISA Kit	Invitrogen	EHCCL13
MicroAmp Fast 96-Well Reaction Plate (0.1 mL)	Applied Biosystems (Waltham, USA)	4346907
MicroAmp Optical Adhesive Film	Life Technologies Corporation (Carlsbad, USA)	4311971
MicroAmp Splash Free 96-Well Base	Applied Biosystems (Waltham, USA)	4312063
Microlitercentrifuge CD-3124R	Phoenix Instrument (Germany)	9013111121
Microscope Cover Glasses, 10 mm	Carl Roth GmbH, Karlsruhe, Germany	4HX4.1
Monocyte Attachment Medium	PromoCell (Heidelberg, Germany)	C-28051
Multiply-Pro Gefäß 0.5 mL, PP	Sarstedt AG & CO (Nümbrecht, Germany)	7,27,35,100
Nanodrop One	Thermo Scientific (USA)	ND-ONE-W
QuantStudio 3 System	Life Technologies GmbH (St. Leon-Rot, Germany)	A28567
RNeasy Micro Kit	Qiagen	74007
RPMI 1640, 1x, with L-glutamine	Mediatech, Inc. (Manassas, USA)	10-040-CV
Sterile bench, LaminarAir HB 2472	Heraeus instruments (Hanau, Germany)	51012197
Tissue Culture Coverslips 13 mm (Plastic)	Sarstedt Inc. (Newton, USA)	83,18,40,002
Titanium machinied discs 12 cm	Medentis Medical (Bad-Neuenahr-Ahrweiler, Germany)	N/A
TNF alpha Human ELISA Kit	Invitrogen	KHC3011
Trypan blue solution 0.4%	Carl Roth GmbH + Co. KG (Karlsruhe, Germany)	1680.1