In Vitro Differentiation of Human Dendritic Cells and their Markers in Leishmania Infection

Summary

Dendritic cells (DCs) are essential components of innate immunity against Leishmania infection. The mechanisms underlying the complex interaction between DCs and Leishmania remain poorly understood. Here, we describe methods to evaluate how Leishmania infection affects the immunobiological function of human DCs, such as migration-related and costimulatory molecule expression.

Abstract

Leishmaniasis comprises a collection of clinical manifestations associated with the infection of obligate intracellular protozoans, Leishmania. The life cycle of Leishmania parasites consists of two alternating life stages (amastigotes and promastigotes), during which parasites reside within either arthropod vectors or vertebrate hosts, respectively. Notably, the complex interactions between Leishmania parasites and several cells of the immune system largely influence the outcome of infection. Importantly, although macrophages are known to be the main host niche for Leishmania replication, parasites are also phagocytosed by other innate immune cells, such as neutrophils and dendritic cells (DCs).

DCs play a major role in bridging the innate and adaptive branches of immunity and thus orchestrate immune responses against a wide range of pathogens. The mechanisms by which Leishmania and DCs interact remain unclear and involve aspects of pathogen capture, the dynamics of DC maturation and activation, DC migration to draining lymph node (dLNs), and antigen presentation to T cells. Although a large body of studies support the notion that DCs play a dual role in modulating immune responses against Leishmania, the participation of these cells in susceptibility or resistance to Leishmania remains poorly understood. After infection, DCs undergo a maturation process associated with the upregulation of surface major histocompatibility complex (MHC) II, in addition to costimulatory molecules (namely, CD40, CD80, and CD86).

Understanding the role of DCs in infection outcome is crucial to developing therapeutic and prophylactic strategies to modulate the immune response against Leishmania. This paper describes a method for the characterization of Leishmania-DC interaction. This detailed protocol provides guidance throughout the steps of DC differentiation, the characterization of cell surface molecules, and infection protocols, allowing scientists to investigate DC response to Leishmania infection and gain insight into the roles played by these cells in the course of infection.

Introduction

Leishmaniasis constitutes a complex of neglected diseases caused by different species of the Leishmania genus¹. Leishmania is an intracellular protozoan of the Trypanosomatidae family that infects humans and other mammals, causing a spectrum of diseases ranging from skin lesions to visceral forms². The main clinical manifestations of this disease are tegumentary leishmaniasis (TL) and visceral leishmaniasis (VL). The World Health Organization (WHO) estimates that 700,000 to 1 million new cases occur annually, causing 70,000 deaths each year². Worldwide, leishmaniasis affects approximately 12 to 15 million people, and 350 million are at risk of contracting the disease³.

The genus Leishmania presents two evolutionary forms: the promastigote and the amastigote⁴. Leishmania promastigotes are characterized by the presence of flagella and high motility. These forms are found in the digestive tract of the sand fly, where they undergo differentiation into the infective form (metacyclic promastigotes)⁵. By contrast, amastigotes are found in the intracellular environment of infected mammalian cells. This evolutionary form, in turn, replicates in the phagolysosomes of phagocytic cells⁶.

The transmission cycle of Leishmania spp. starts during blood-feeding, when sandflies inoculate metacyclic promastigotes into the host's skin¹. Shortly after Leishmania inoculation, innate immune cells, including neutrophils and tissue-resident macrophages, phagocytize the parasites. Inside parasitophorous vacuoles, Leishmania differentiate into amastigotes and replicate, culminating in the rupture of the host cell membrane, which allows the infection of neighboring cells and parasite spread⁴. The cycle is completed when phlebotomines ingest amastigote-containing phagocytes, which differentiate into procyclic promastigotes and later into metacyclic promastigotes in the insect's intestinal tract⁷.

Dendritic cells, professional antigen-presenting cells found in tissues and lymph nodes, act as a sentinel for the immune system⁸. These cells are found in peripheral tissues at immature stages, mainly involved in antigen capture and processing. After contact with pathogens, DCs undergo a maturation process that culminates in their migration to the lymph nodes, subsequently presenting antigens to naïve CD4⁺ T cells. These cells are also essential in orchestrating the innate and adaptative immune responses that generate tolerance or inflammation⁹. The DC maturation process involves several aspects, including increased expression of MHC and costimulatory molecules, such as CD40 and CD86, as well as enhanced cytokine secretion. DCs express different markers, including CD11b and CD11c, and, in humans, the DCs that originate from CD14⁺ monocytes (moDCs) express CD1a¹⁰. CCR7 is highly expressed on DCs and indicates the complex migratory process of these cells¹². CD209 and CD80 also play an important role in the initial contact with DCs and lymphocytes¹³.

In leishmaniasis, studies suggest that moDCs phagocytose parasites and deliver them to the draining lymph nodes (dLNs), where they present antigens to T cells¹³. The parasite capture mechanism is associated with cytoskeletal reorganization by actin filaments during phagocytosis, which promotes the internalization of the parasite¹⁴. Most studies concerning the roles exercised by DCs in leishmaniasis have focused on L. major, L. amazonensis, and L. braziliensis¹⁵. Interestingly, in vivo studies of Leishmania infection have demonstrated that the impairment of DC function occurs in a parasite strain-specific manner.

It has been demonstrated that during the early stages of L. amazonensis infection, DCs exhibit a decreased ability to constrain parasite infection. Conversely, in an experimental model of L. braziliensis infection, DCs were shown to mount appropriate immune responses that restricted Leishmania survival¹⁶. The chief aspects known to be associated with differential responses to Leishmania spp. infection are the degree of DC maturation and activation. This paper describes a method to investigate the role human DCs play in Leishmania infection to further understand how these cells influence disease outcomes.

Protocol

NOTE: Cells were obtained from healthy donor volunteers. The procedure described herein was approved by the National Ethics Committee (number 2.751.345)-Fiocruz, Bahia, Brazil). 1. Differentiation of human dendritic cells Pipette 10 mL of polysucrose-sodium triazoate mixture in 50 mL conical tubes. Label the 50 mL conical tubes respectively for each donor. Collect 30 mL of blood from healthy donors and perform all subsequent steps in the laminar…

Representative Results

This report investigates the role of DCs in Leishmania infection using flow cytometry and confocal microscopy. Initially, the phenotypic profile of the human monocyte-derived DC was established. Notably, the obtained CD11c+ dendritic cell populations were positive for CCR7, CD209, CD80, CD1a, and HLA-DR. The results indicate that the expression of these markers in DC populations is profoundly impacted by Leishmania infection. Infected DCs exhibited augmented CD80, CD209, CCR7, and HLA-DR expr…

Discussion

Leishmaniasis is a severe public health problem worldwide. The pathogenesis of this disease is quite complex, and the mechanisms favoring parasite survival in vertebrate hosts remain elusive¹⁷. DCs are professional antigen-presenting cells found throughout the body, including filtering and lymphoid organs. Following antigen capture and processing, immature DCs undergo a complex maturation process that culminates in their migration to lymph nodes, where these cells are responsible for presenting an…

Materials

anti CCR7	Thermo
anti CD209	Isofarma
anti CD83	Leica SP8
anti HLA-DR	Gibco
Bovine serum albumin	Thermo	A2153-100G	Sigma
Ciprofloxacin	Gibco
confocal microscope	Thermo Fisher Scientific
Fetal bovine serum	Gibco
Flow Jo	Thermo Fisher Scientific
Gentamicin	Thermo Fisher Scientific
Glutamin	Gibco
HEPES	Thermo Fisher Scientific
phalloidin	Thermo Fisher Scientific
Phosphate buffer solution	Peprotech
prolong gold antifade kit	BD pharmigen
RPMI	BD pharmigen
Saponin	BD pharmigen	47036 – 50G – F	Sigma
Schneider's insect medium	software BD biosciences