Elsevier

Immunology Letters

Volume 201, September 2018, Pages 20-30
Immunology Letters

Impact of the GK-1 adjuvant on peritoneal macrophages gene expression and phagocytosis

https://doi.org/10.1016/j.imlet.2018.10.010Get rights and content

Highlights

Abstract

Purpose

The synthetic peptide GK-1 potentiates protective immunity elicited by the influenza vaccine in mice. In order to understand its adjuvant properties, this study was designed to determine the impact of GK-1 on gene expression and phagocytosis of peritoneal macrophages (PMa).

Methods

Increased gene expression of chemokines involved in leukocyte recruitment and of pro-inflammatory mediators was detected by microarray analysis of control and GK-1 treated PMa macrophages. The expression profile was subsequently confirmed by Multiplex Immunoassays analysis to measure cytokines levels, flow cytometer to describe M1/M2 surface markers and an assay to evaluate their phagocytic activity.

Results

Treatment of PMa with GK-1 results in development to the classically activated M1 functional macrophage subpopulation with increased expression of the CCL3 and CXCLO2 chemokines, IL-6 and TNF-α proinflammatory cytokines with a concomitant increase in the levels of NO, accompanied by the expression of modulatory factors that downregulate the inflammatory phenotype. GK-1 treated PMa significantly increased their phagocytic activity.

Conclusion

GK-1 classical activated with enhanced phagocitic capacity may underlie in the increased specific immunity induced when concomitant administered with other antigens.

Introduction

Despite the clear positive impact of vaccination on global protection against infectious diseases, there are an estimated 44 million cases of life threatening diseases occurring in 219 nations. The development of new tools to improve vaccine efficiency is one realistic alternative to deal with the magnitude of this task. Indeed, a better understanding of the relevance of innate immunity in controlling the acquired immune response offers rational approaches for immunomodulation in order to improve the limited efficiency and undesirable effects of the available adjuvants [1].

Adjuvants may act at different levels of the immune system. Depending on their biochemical nature, they can selectively inhibit or enhance the functional differentiation of the different cell populations comprising the immune system (i.e. lymphocytes, neutrophils, NK killer cells, dendritic cells and macrophages) [1,2]. Between them, macrophages play a pivotal role in activating and directing both innate and acquired immunity through their variable cytokine responses to different modes of antigen presentation. These, together with cytokines and chemokines secreted by other cells early in the innate immune response, not only affect monocytes at sites of infection, but also create the cytokine “milieu” that guides the differentiation of monocytes to macrophages and similarly dictates the functional activities of existing macrophages. Their high functional heterogeneity and plasticity highlights macrophages and monocytes as a pivotal target for modulation by adjuvants.

Two major macrophage phenotypes derived from monocytes have been operationally defined, the classically activated M1 and the alternatively activated M2 macrophages. The M1 cells are induced by IFNץ and/or TNFα or bacterial lipopolysaccharide (LPS) and have the following properties: the killing of intracellular parasites and tumor cells, the secretion of pro-inflammatory cytokines and chemokines, the production of nitric oxide (NO), an increased expression of MHC class II, CD80, CD86 molecules, and antigen presentation associated with Th1 cells [3,4]. In contrast, the M2 alternative activated macrophages are involved in the resolution of inflammation and tissue homeostasis and have been further subdivided into at least three different overlapping sub-phenotypes (M2a, b, c), a classification which will almost certainly be revised, given more information; for example, it has been observed that the phenotype of macrophages activated through IL-4 overlap partially with those induced by IL-13, IL-10, TGFβ and glucocorticoids [5,6]. Similarly, human monocytes differentiated to macrophages in the presence of IL12 and IL18 are distinct from either M1 or M2 phenotypes [7].

Peritoneal macrophages, cells generated and maturated within the body in a dynamic compartment as the peritoneum offer an accurate tool to study ex vivo, the effect of GK-1 [8]. In this study, therefore, we focus on the impact of GK-1 on murine peritoneal macrophages. This peptide, belongs to the Taenia crassiceps cysticercus recombinant protein KETc7 [9] and has being shown to enhance the protection induced by human influenza vaccine in both young and old mice [10,11]. In addition, GK-1 used as adjuvant provided some degree of protection against experimental murine melanoma and mammary cancers [12]. Perhaps relevant to its adjuvant properties GK-1 promoted the activation of dendritic cells, increasing the expression of CD80/86, MHC class II, and the secretion of IFN-γ, TNF-α, and CCL2 (MCP-1), resulting in an increase in CD4 + T cell proliferation [10,13]. Considering that the plasticity of macrophages can result in awake the adaptative immunity, we extend our studies evaluating GK-1 effect on in vivo matured peritoneal macrophages towards the identification of other possible players that may underlie GK-1 adjuvant properties.

Section snippets

Mice

BALB/cAnN female mice 6–8 weeks-old were used. Stocks were originally purchased from Charles River, USA, and produced in a pathogen-free facility at our Institute. Food and water were allowed ad libitum. All housing and experimental procedures were conducted under the guidelines established by the Committee on the Care and Use of Experimental Animals of the Instituto de Investigaciones Biomédicas (IBM) at the Universidad Nacional Autónoma de México (UNAM). Mouse experimentation protocols were

GK-1 induced differential gene expression of peritoneal macrophages

A detailed transcriptional profile of peritoneal macrophages treated with GK-1 at 4 h at 18 h was subjected to gene expression profiling analysis with whole genome microarrays (Fig. 1A, B). Unsupervised hierarchical clustering analysis showed a differential expression between control and GK-1 treated macrophages that allowed us to perform the comparison between the heat-maps resulting from each exposure time of 4 and 18 h to GK-1.

Of the total of 28,944 genes included in the microarray, 503 were

Discussion

Macrophages play a key role in tissue homeostasis, inflammation, immunity, and disease pathogenesis. The wide spectrum of macrophage activities is reflected in an equally complex range of functionally distinct populations differentiated from monocytes according to the prevailing cytokine “milieu”, created as a result of the early inflammatory response to a pathogen.

Historically, those macrophages differentiated in the presence of IFNγ, LPS, or GMCSF were defined as the M1 inflammatory

Ethical approval

All applicable international, national, and/or institutional guidelines for the care and use of animals were followed.

This article does not contain any studies with human participants performed by any of the authors.

Conflict of interest

The authors declare that they have no conflict of interest.

Acknowledgments

This work was supported by CONACyT project No. 253891 and the Program of Research for the Development and Optimization of Vaccines, Immunomodulators and Diagnostic Methods of the Biomedical Research Institute from National Autonomous University of Mexico (UNAM).

Lisandro Sánchez Hernandez PhD. Biomedical sciences student Biomedical research institute National Autonomous University of Mexico, UNAM Work areas Development of adjuvants.

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  • Cited by (0)

    Lisandro Sánchez Hernandez PhD. Biomedical sciences student Biomedical research institute National Autonomous University of Mexico, UNAM Work areas Development of adjuvants.

    Laura Montero Leon PhD. Biomedical sciences student Biomedical research institute National Autonomous University of Mexico, UNAM Work areas Development of adjuvants

    Raúl Mojica Espinosa MSc. Ascription: Subdirection of development of clinical applications Position: Head of Department in Medical Area B National Institute of Genomic Medicine of Mexico

    Juan Pablo Reyes Grajeda PhD. PhD. Biomedical sciences National Autonomous University of Mexico, UNAM Associate Researcher in the Medical Proteomics Unit National Institute of Genomic Medicine of Mexico Work areas Expert in the purification and biochemical characterization of proteins, as well as in crystallogenesis and crystallography.

    Jacquelynne Cervantes Torres PhD. Biomedical sciences student Biomedical research institute National Autonomous University of Mexico, UNAM Work areas Development of adjuvants, expert in vitro and in vivo testing of several biological molecules involved in cell differentiation and activation.

    Michael Parkhouse PhD. Biochemistry University of London, United Kingdom (UK) Researcher of the Department of Infections & Immunity Gulbenkian Institute of Science Work areas Infectious organism and its host

    Gladis Fragoso Gonzalez PhD. Researcher of Department of Immunology Biomedical Research Institute National Autonomous University of Mexico, UNAM Work areas Immunoparasitology. Neuroinflammation Identification of antigens and protective epitopes against cysticercosis.Diagnosis of human and porcine cysticercosis Development of adjuvants

    Edda Lydia Sciutto Conde PhD. Researcher of Department of Immunology Biomedical Research Institute National Autonomous University of Mexico, UNAM Work areas Immunoparasitology. Neuroinflammation.Identification of antigens and protective epitopes against cysticercosis.Diagnosis of human and porcine cysticercosis Development of adjuvants

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