Actives from the Micro-Immunotherapy Medicine 2LMIREG^® Reduce the Expression of Cytokines and Immune-Related Markers Including Interleukin-2 and HLA-II While Modulating Oxidative Stress and Mitochondrial Function

Abstract

Introduction

Micro-immunotherapy (MI) is a therapeutic option employing low doses (LD) and ultra-low doses (ULD) of cytokines and immune factors to help the organism at modulating the immune responses. In an overpowering inflammatory context, this strategy may support the restoration of the body’s homeostasis, as the active ingredients of MI medicines’ (MIM) could boost or slow down the physiological functions of the immune cells. The aim of the study is to evaluate for the first time the in vitro anti-inflammatory properties of some actives employed by the MIM of interest in several human immune cell models.

Methods

In the first part of the study, the effects of the actives from the MIM of interest were assessed from a molecular standpoint: the expression of HLA-II, interleukin (IL)-2, and the secretion of several other cytokines were evaluated. In addition, as mitochondrial metabolism is also involved in the inflammatory processes, the second part of the study aimed at assessing the effects of these actives on the mitochondrial reactive oxygen species (ROS) production and on the mitochondrial membrane potential.

Results

We showed that the tested actives decreased the expression of HLA-DR and HLA-DP in IFN-γ-stimulated endothelial cells and in LPS-treated-M1-macrophages. The tested MIM slightly reduced the intracellular expression of IL-2 in CD4⁺ and CD8⁺ T-cells isolated from PMA/Iono-stimulated human PBMCs. Additionally, while the secretion of IL-2, IL-10, and IFN-γ was diminished, the treatment increased IL-6, IL-9, and IL-17A, which may correspond to a “Th17-like” secretory pattern. Interestingly, in PMA/Iono-treated PBMCs, we reported that the treatment reduced the ROS production in B-cells. Finally, in PMA/Iono-treated human macrophages, we showed that the treatment slightly protected the cells from early cell death/apoptosis.

Discussion

Overall, these results provide data about the molecular and functional anti-inflammatory effects of several actives contained in the tested MIM in immune-related cells, and their impact on two mitochondria-related processes.

Keywords:

• micro-immunotherapy
• mitochondrial metabolism
• cytokines
• inflammation
• anti-inflammatory
• interleukin-2

Abbreviations

ATP, adenosine triphosphate; BHA, butylated hydroxyanisole; BSA, bovine serum albumin; CH, centesimal Hahnemannian; CXCL10, C-X-C motif ligand 10; DHR, dihydrorhodamine; DNA, deoxyribonucleic acid; ECBM, endothelial cell growth medium; EFS, Etablissement Français du Sang; ELISA, enzyme-linked immunosorbent assay; FBS, fetal bovine serum; FSC, forward scatter; HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; HLA, human leukocyte antigen; hr, human recombinant; HUVEC, human umbilical vein endothelial cells; GMFI, geometric mean fluorescence intensity; IFN-γ, interferon-γ; IL, interleukin; Iono, Ionomycin; JC-10, 5.6-dichloro-1,1’,3,3’-tetraethylimidacarbocyanine iodide; LD, low doses; LPS, lipopolysaccharide; M-CSF, macrophage colony-stimulating factor; MFI, median fluorescence intensity; MI, micro-immunotherapy; MIM, micro-immunotherapy medicine; mtMP, mitochondrial membrane potential; PBMCs, peripheral blood mononuclear cells; PBS, phosphate-buffered saline; PGE2, prostaglandin E2; PMA, phorbol myristate acetate; RNA, ribonucleic acid; ROS, reactive oxygen species; RPMI, Roswell Park Memorial Institute medium; S.D., standard deviation; S.E.M., standard error of the mean; SNA^®, specific nucleic acids; SNs, supernatants; SSC, side scatter; TGF-β, tumor growth factor-β; TLR, Toll-like receptor; TNF-α, tumor necrosis factor-α; ULD, ultra-low doses; Veh., vehicle.

Acknowledgments

The authors are grateful to the staff of QIMA Sciences, especially Adrien Brulefert for their work in performing the experiments of the in vitro study on PMA-stimulated granulocytes, and Laura Garcia-Sureda, from Labo’Life Spain, for having managed this part of the project. The authors would like to thank Miquel Ensenyat and his team for having prepared, provided and sent the Veh. and the tested MIMs to ProfileHIT and QIMA facilities. The authors thank Sofia Frau for the insightful comments that improved the final version of the manuscript. Finally, the authors thank Servier Medical Art (https://smart.servier.com/, accessed on the 02/06/2023) for the items used in the figures.

Disclosure

The authors declared the following conflicts of interest with respect to the research, authorship, and/or publication of this article: Camille Jacques and Ilaria Floris work for Labo’Life France, the company service provider of Labo’Life, specialized in preclinical research and regulatory affairs. This professional relationship does not imply any misconduct on the part of the authors. Mathias Chatelais and Flora Marchand work for ProfileHIT, an innovative profiling company involved in vascular and immunology crosstalk research field in human. This study was entirely funded by Labolife France. The authors report no other conflicts of interest in this work.