Fluorescence Assays for the Study of Mycobacterium tuberculosis Interaction with the Immune Receptor SLAMF1

Summary

This study provides a protocol for evaluating the interaction of Mycobacterium tuberculosis with the SLAMF1 microbial sensor. The assays were conducted on human monocyte-derived macrophages using flow cytometry and fluorescence microscopy. The described tools are relevant for studying interactions between pathogens and immunoreceptors.

Abstract

The evaluation of direct interaction between pathogens and immune receptors usually involves sophisticated techniques or implies the use of transgenic strains and genetically engineered cells. Here, an alternative method to detect biochemical interaction between the macrophage microbial sensor SLAMF1 and Mycobacterium tuberculosis is described. Two technical approaches employing flow cytometry and fluorescence microscopy were developed. Total cell protein extracts from human macrophages were generated, then incubated with whole cells of M. tuberculosis (WCMtb) or M. tuberculosis antigens (Mtb Ags) overnight at 4 °C and finally cross-linked using formaldehyde/glycine/ethylene glycol bis (succinimidyl succinate) treatment. SLAMF1 interaction with WCMtb by flow cytometry was detected with a PE-specific anti-SLAMF1 antibody. The existence of interaction by fluorescence microscopy was performed by attaching Rhodamine-PE stained Mtb Ags to poly-D-lysine coated slides, which were incubated with the total protein extract from monocyte-derived macrophages. After cross-linking treatment, SLAMF1 was visualized using primary (anti-SLAMF1) and secondary (Alexa Fluor 488) antibodies. The assays provided a strong biochemical tool to measure pathogen-immunoreceptor interactions, overcoming the difficulties associated with transgenic cell lines and protein gene expression modulation experiments.

Introduction

Mycobacterium tuberculosis, the Tuberculosis-causative pathogen identified 142 years ago, remains a global challenge, currently infecting at least a quarter of the world's population¹. Transmitted through airborne droplets from infected people, M. tuberculosis reaches alveolar macrophages in the lungs where it can survive for long periods in a latent state^2,3. Not only is the local macrophagic response activated, but in recent years, it has been described that peripheral monocytes can be recruited to the respiratory tract and differentiate into alveolar macrophages

Protocol

All procedures involving human monocyte-derived macrophages were performed in accordance with the Helsinki Declaration (2013) and in agreement with the Ethics Committee of UNNOBA (COENOBA). Written informed consent was obtained prior to sample collection. The male/female group distribution was 13/6, and the median age was 32 years, with an interquartile range (IQR) of 18-75 years. The presence of previous pathologies, comorbidities, or a positive diagnosis for Tuberculosis were defined as exclusion criteria. The details of the reagents and equipment are listed in the Table of Materials.

1. Monocyte-derived macrophage

Results

In this work, a protocol that allows the evaluation of M. tuberculosis interaction with the immune receptor SLAMF1 in human macrophages is provided (Figure 1). To this end, peripheral blood from healthy donors was obtained. Then, PBMCs were separated by centrifugation over the density gradient media, and the monocytes were isolated by magnetic positive selection (≥95% purity, Figure 2A,B). The isolated monocytes were adhered to pl...

Discussion

This study provides a useful guide for studying the biochemical interaction between M. tuberculosis and microbial sensors expressed in human macrophages, a key cell type involved in the host response during Tuberculosis. The provided protocols will be relevant to decipher molecules that play a role in the entry of M. tuberculosis into phagocytes.

Characterizing bio-molecular interactions, such as that between pathogens and immunoreceptors, is crucial to understanding both imm...

Materials

Name	Company	Catalog Number	Comments
Alexa Fluor 488 secondary antibody	Invitrogen	A21121	For fluorescence microscopy
anti-SLAMF1 FITC antibody	eBioscience	11-1509-42	For flow cytometry
anti-SLAMF1 PE antibody	BioLegend	306308	For flow cytometry
anti-SLAMF1 primary antibody	BioLegend	306302	For fluorescence microscopy
Aqua-Poly/Mount	Polysciences	18606-20	Mounting media
CD14 MicroBeads	Miltenyi Biotec	130-097-052	For monocytes isolation
Coverslips 12mm	HDA	-	For interaction assay by microscopy
EGS	ThermoFisher Scientific	21565	For crosslinking treatment
FACSCanto II	BD Biosciences	338960	Flow cytometer with BD FACSDiva software
Fetal Bovine Serum	Natocor	-	Inactivated and irradiated, for macrophages culture
Ficoll-Paque PLUS	Cytiva	17144003	For PBMCs separation
Fiji/ImageJ	Open Source software	-	For micrographs analysis
FlowJo 7.6.2	Tree Star	-	For flow cytometry analysis
Formaldehyde	Merck	K47740803613	For crosslinking treatment
Glass slides	Glass Klass	-	For interaction assay by microscopy
Glycine	Sigma	G8898	For crosslinking treatment
Imager.A2	Carl Zeiss	430005-9901-000	Fluorescence microscope with Colibri 7 illumination module
iMark	BIO-RAD	1681130	Microplate absorbance reader
L-glutamine	Sigma Aldrich	49419	For macrophages culture
M. tuberculosis, strain H37Rv, gamma-irradiated whole cells	BEI Resources, NIAID, NIH	NR-14819	For interaction assay
M. tuberculosis, strain H37Rv, whole cell lysate	BEI Resources, NIAID, NIH	NR-14822	For macrophages stimulation and interaction assay
Neofuge 13R	Heal Force	Neofuge 13R	High Speed Refrigerated Centrifuge for protein extraction
Penicillin/Streptomycin	Gibco	15140122	For macrophages culture
PMSF	ThermoFisher Scientific	36978	For proteins isolation
Poly-D-Lysine	Sigma Aldrich	A-003-M	For coverslips treatment
Protease Inhibitor Cocktail	Sigma Aldrich	P8340	For proteins isolation
Rhodamine B	Sigma Aldrich	21955	For M. tuberculosis staining
RPMI 1640	Gibco	11875093	For macrophages culture
Sorvall ST 16/16R centrifuge	ThermoFisher Scientific	75004240	For PBMCs and monocytes isolation