Lentiviral Vector Preparation for Efficient Gene and MicroRNA Modulation of Peritoneal Cavity Tissue-Resident Macrophages In Vivo in Mice

Summary

We demonstrate a step-by-step protocol for the investigation of gene function in peritoneal tissue-resident macrophages in vivo, using lentiviral vectors.

Abstract

Peritoneal tissue-resident macrophages have broad functions in the maintenance of homeostasis and are involved in pathologies within local and neighboring tissues. Their functions are dictated by microenvironmental cues; thus, it is essential to investigate their behavior in an in vivo physiological niche. Currently, specific peritoneal macrophage-targeting methodologies employ whole-mouse transgenic models. Here, a protocol for effective in vivo modulation of mRNA and small RNA species (e.g., microRNA) expression in peritoneal macrophages using lentivirus particles is described. Lentivirus preparations were made in HEK293T cells and purified on a single sucrose layer. In vivo validation of lentivirus effectivity following intraperitoneal injection revealed predominant infection of macrophages restricted to local tissue. Targeting of peritoneal macrophages was successful during homeostasis and thioglycolate-induced peritonitis. The limitations of the protocol, including low-level inflammation induced by intraperitoneal delivery of lentivirus and time restrictions for potential experiments, are discussed. Overall, this study presents a quick and accessible protocol for the rapid assessment of gene function in peritoneal macrophages in vivo.

Introduction

Tissue-resident macrophages (Mφ) are a heterogeneous population of phagocytic immune cells that sense and respond to invading pathogens^1,2. In addition, they play an essential role in tissue development, remodeling, and maintaining homeostasis^1,3. Many tissue Mφ derive from yolk sac progenitors during embryogenesis and persist in the tissue throughout the life^4,5. The phenotype and functions of these cells are dictated by collaborative and hierarchical interactions of specific transcr....

Protocol

All animal work was conducted in accordance with Institutional and UK Home Office guidelines.

NOTE: All in vivo studies with lentivirus should be performed according to local and national guidelines on the ethical use of animals in research, as well as adhering to all regulations associated with the use of category II infectious materials. Animal welfare should also be monitored in accordance with local regulations. In this step of the protocol, extreme care needs to be taken when wor.......

Discussion

Tissue-resident macrophages perform a range of homeostatic and inflammatory tissue-specific functions^1,2 dictated by their physiological environment^6,7,8,9. In this protocol, an effective method¹⁸ for manipulation of peritoneal resident macrophages in vivo using lentivirus particles was introduced to inv.......

Materials

Name	Company	Catalog Number	Comments
0.05% Trypsin-EDTA (1x) (Trypsin 500 mg/L or 0.02 mM)	Thermo Fisher Scientific	25300054
0.22 μm sterile millex GP filter	Merck	SLGS033SS
0.45 μm sterile millex GP filter	Merck	SLHP033RS
0.5 mL U-100 insulin syringe with needle, 0.33 mm x 12.7 mm (29 G)	BD	324892
1 Litre Sharps Container	N/A	N/A
2.4G2 antibody (TruStain FcX anti-mouse CD16/32)	Biolegend	101320
40 μm strainer	Thermo Fisher Scientific	22363547
AimV medium (research grade), AlbuMax Supplement	Thermo Fisher Scientific	31035025
Blocking buffer	prepared in house
Brewer thioglycolate medium	Sigma-Aldrich	B2551	4% stock solution prepared in water, autoclaved and kept frozen.
CD11b	Biolegend	101222	Refer to Table 1 for the dilution and concentration
CD11b	BD	550993	Refer to Table 1 for the dilution and concentration
CD11c	Biolegend	117317	Refer to Table 1 for the dilution and concentration
CD11c	Biolegend	117333	Refer to Table 1 for the dilution and concentration
CD19	BD	560375	Refer to Table 1 for the dilution and concentration
CD19	Biolegend	152410	Refer to Table 1 for the dilution and concentration
CD226	Biolegend	128808	Refer to Table 1 for the dilution and concentration
CD3e	BD	560527	Refer to Table 1 for the dilution and concentration
CD3e	Biolegend	152313	Refer to Table 1 for the dilution and concentration
CD4	Biolegend	100412	Refer to Table 1 for the dilution and concentration
CD73	eBioscience	16-0731-82	Refer to Table 1 for the dilution and concentration
CD8a	eBioscience	48-0081-82	Refer to Table 1 for the dilution and concentration
Cell culture flask (T175 fask, 175 cm2, 550 mL)	Greiner Bio One	658175
Centrifuge tubes, conical bottom tubes 25 mm x 89 mm	Beckman Coulter	358126
Centrifuges	Beckman Coulter		Ultracentrifuge and TC centrifuge
Collagenase type IV	Sigma-Aldrich	C5138
Conical centrifuge tubes (15 mL and 50 mL)	Greiner Bio One	11512303 & 11849650
Cryotubes	Greiner Bio One	123277	or cryotubes
DMEM medium (1x) + 4.5g/L D-glucose, 400 µM L-glutamine	Thermo Fisher Scientific	41965-062
Dnase I	Sigma-Aldrich	11284932001
Effectene transfection reagent	Qiagen	301425
F4/80	Biolegend	123123	Refer to Table 1 for the dilution and concentration
F4/80	Biolegend	123133	Refer to Table 1 for the dilution and concentration
F4/80	Biolegend	123147	Refer to Table 1 for the dilution and concentration
FceR1	eBioscience	48-5898-80	Refer to Table 1 for the dilution and concentration
Fetal calf serum (FCS)	Thermo Fisher Scientific	10270-106	heat inactivated for 30 min at 56 °C and sterile filtered through 0.22 μm filter
Flow cytometer	Thermo Fisher Scientific	Attune NxT
Flow cytometry (FACS) buffer	prepared in house
Fluorescent tissue culture microscope	Thermo Fisher Scientific	EVOS FL
Forceps	N/A	N/A	User preference
Hank's balanced salt solution (HBSS)	Gibco, Life Technologies	14175-053
HEK293T cell line			grown for at least a week prior transfection. Mycoplasma free
HIV-1 Core antigen	Beckman Coulter	6604667
Hyaluronidase	Sigma-Aldrich	H3506
Hydrex surgical scrub, chlorhexiding gluconate 4% w/v skin cleanser	Ecolab	3037170
I-A/I-E	Biolegend	107625	Refer to Table 1 for the dilution and concentration
ICAM1	Becton Dickinson	554970	Refer to Table 1 for the dilution and concentration
Jurkat T cell line			grown for at least a week prior use. Mycoplasma free
LIVE/DEAD fixable near-IR dead cell stain kit	Thermo Fisher Scientific	L34975
Ly6G	Biolegend	127615	Refer to Table 1 for the dilution and concentration
Mice			here used C57BL/6 females, aged 8-12 weeks (Charles Rivers), unless specified differently
Microcapillary pipettes (volume range 0.5-1,000 μL)	Fisher Scientific & Starlab	11963466 & 11943466 & 11973466 & S1111-3700
NK1.1	Biolegend	108724	Refer to Table 1 for the dilution and concentration
Paraformaldehyde	Sigma-Aldrich	P6148-500G	prepared to 2% w/v in PBS
pCMV-ΔR8.91 packaging plasmid		Zuffrey, R., et al. 1997	encodes Gag-Pol HIV protein driven by cytomegalovirus promoter. Ampicilin resistance.
Penicillin/Streptomycin (100x, 10,000 U/mL)	Thermo Fisher Scientific	15140122
Petri dish	Greiner Bio One	664160
pHR'SIN-cPPT-SEW plasmid		Rosas, M. et al. 2014	modified for shRNA and miR expression studies. Encodes EGFP marker downstream SFFV promoter and upstream of the Woodchuck hepatitiv virus enhancer. Ampicilin resistance.
pMD2.G plasmid		Naldini, L. et al. 1996	encodes vesicular stomatis virus g-glycoprotein (VSV-G) envelope. Ampicilin resistance.
Rat IgG1, κ isotype control	Becton Dickinson	550617	Refer to Table 1 for the dilution and concentration
Rat serum	Sigma-Aldrich	R9759-10ML
Red blood ACK lysis buffer	prepared in house
RPMI 1640 medium (1x) + 400 uM L-glutamine	Thermo Fisher Scientific	21875-091
Saponin	Sigma-Aldrich	S4521
SiglecF	BD	562681	Refer to Table 1 for the dilution and concentration
Sodium hypochlorite Tablets (bleach, 2,000 ppm)	Guest Medical	H8818
Sterile 24-well cell culture plate	Greiner Bio One	662160
Sterile Dulbecco's PBS (DPBS) (1x) Mg++ and Ca2+ - free	Thermo Fisher Scientific	14190144
Sterile EDTA	Thermo Fisher Scientific	15575020
Sterile VWR disposable transfer pipets (23.0 mL, 30 cm)	VWR	612-4515
Sucrose	Thermo Fisher Scientific	15503022
surgical scissors	N/A	N/A	User preference
Syringes (50 mL and 1 0mL)	Fisher Scientific	10084450 & 768160
Tim4	Biolegend	130007	Refer to Table 1 for the dilution and concentration
U-bottom 96-well cell culture plate	Greiner Bio One	650180