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: anti-inflammatory; cytokines; inflammation; interleukin-2; micro-immunotherapy; mitochondrial metabolism.

Figure 1

Actives from the micro-immunotherapy medicine MIM-7 reduce the expression of HLA-II in HUVEC cells and in human PBMCs-derived M1-macrophages. (A) HUVEC cells were treated with 20 ng/mL IFN-γ for 48 hours and the expression of HLA-DR was assessed by flow cytometry. The results are presented as the mean ± standard deviation (S.D.) of the median of fluorescence of n = 3 replicates. (B) HUVEC cells were treated with 20 ng/mL IFN-γ, concomitantly with either the Veh. (grey histogram), or MIM-7 (purple histograms) for 48 hours and the expression of HLA-DR was assessed by flow cytometry. The effects of two independent capsules of MIM-7 (#1 and #2) were assessed. The results are presented as the mean ± S.D. of the percentage of the median fluorescence of the Veh., for n = 3 replicates. (C) Representative scheme of the experimental protocol. Human PBMCs from two healthy donors were seeded at 500.000 cells/well in complete medium added with 2% decomplemented human serum and 50 ng/mL macrophage colony-stimulating factor (M-CSF) to promote macrophage survival. On day 0 (D0), the cells were treated either with the Veh., or MIM-7, added with 20 ng/mL IFN-γ. The medium/treatment was renewed on D3, and the cells were challenged with 100 ng/mL LPS on D5. On D6, the cells were analyzed by flow cytometry for their viability and their expression levels of HLA-DP. LPS: lipopolysaccharide; RPMI: Roswell Park Memorial Institute medium. (D) Cell viability of M1-macrophages isolated from two healthy donors, treated for 6 days with either the Veh. (grey dots), or MIM-7 (purple dots), in the presence of IFN-γ (20 ng/mL) and LPS (100 ng/mL). The data are presented as the mean ± standard error of the mean (S.E.M.) of the percentage of viable cells amongst the total cells, for n = 3 replicates per donor. (E) Expression of the membrane-marker HLA-DP in M1-macrophages isolated and treated as described in (C). The data are presented as the mean ± S.E.M. of HLA-DP expression for n = 3 replicates per donor. The results are displayed as percentages of the Veh. condition for the two individual donors analyzed. The dotted lines in (D and E) highlight the effect of MIM-7 compared with the Veh.

Figure 2

Actives from the micro-immunotherapy medicine MIM-10 slightly reduce the intracellular expression of IL-2 in PMA/Iono-stimulated human lymphocytes. (A and B) PBMCs from four healthy donors were treated for 16 hours with 1X PMA/Iono and the intracellular expression of IL-2 was assessed by flow cytometry, after permeabilization and immune-staining with an anti-IL-2 antibody. The results are presented within the CD4⁺ T-cells and the CD8⁺ T-cells. (C and D) PBMCs from four healthy donors were treated either with the Veh., or with MIM-10 for 48 hours, and were concomitantly stimulated with PMA/Iono during the last 16 hours of incubation. The data are presented as the mean ± S.E.M. of IL-2 expression for n = 3 replicates per donor. The results are displayed as percentages of the Veh. condition for the four individual donors. The dotted lines in (C and D) highlight the effect of MIM-10 compared with the Veh.

Figure 3

Actives from MIM-10 modulate the cytokine-secretion profile of PMA/Iono stimulated-PBMCs. PBMCs were treated for 48 hours with either the Veh. (grey histograms) or MIM-10 (purple histograms) and a PMA/Iono treatment was applied during the last 16 hours as a pro-inflammatory stimulus. The SN were harvested and the cytokine content was appraised by ELISA method. (A–F) The secretion of IL-2, IL-10, IFN-γ, IL-6, IL-9 and IL-17A was measured. The data are presented as the mean ± S.E.M. of n = 4 donors. The results are displayed as percentages of the Veh. condition for each of the four individual donors, each dot representing the mean value obtained for one donor (n = 3 replicates). The dotted lines highlight the effect of MIM-10 compared with the Veh.

Figure 4

Commercially available rh IL-1β, IL-2, TGF-β and TNF-α are proper inducers of mitochondrial-ROS production in lymphocytes and neutrophils isolated from human PBMCs. Briefly, PBMCs from 3 donors were isolated and left untreated (Ct.) or incubated for 24 hours in the presence of 20 ng/mL of either IL-1β, IL-2, TGF-β, or TNF-α. At the end of the incubation period, the cells were stained with MitoROS^TM 580 and analyzed by flow cytometry. The ROS signal was discriminated between: (A) the lymphocytes and (B), the neutrophils, based on their forward scatter/side scatter (FSC/SSC). The ROS signals’ results are presented as the mean percentage ± S.D. of the detected signal (median fluorescence intensity [MFI]), each dot representing the data obtained for one donor. The dotted lines highlight the basal level of ROS in the Ct. conditions.

Figure 5

Actives from MIM reduce the mitochondria-specific ROS production in human PBMCs-derived B-cells and neutrophils. The cells were left untreated (Ct.) or were stimulated with 1X PMA/Iono for one hour, stained with MitoROS^TM 580, and incubated with a panel of antibodies allowing for the discrimination of the different immune-cell sub-populations (B-cells and neutrophils). (A and B) The production of ROS was assessed by flow cytometry, and is expressed as the MFI of the MitoROS^TM 580 signal, measured in the PBMCs from n = 4 donors. The results are expressed as the mean MFI ± S.E.M. of the values obtained for n = 3 measures per donor, the Ct. being a triplicate measure of a pool of the four donors. (C–E) The cells were treated for 48 hours with either the Veh, or the MIM capsules before PMA/Iono stimulation and immune-staining. (C) The cell viability of the whole PBMCs was appraised by flow cytometry, and is expressed as a percentage of the live cells amongst the total cells, for the four analyzed donors. (D and E) The ROS production was assessed by flow cytometry, within the B-cells and the neutrophils sub-populations. The results are expressed as the mean MFI ± S.E.M. of the values obtained for n = 3 measures per donor. The dotted lines in (D and E) highlight the effect of MIM capsules compared with the Veh.

Figure 6

Actives from MIM modulate the mtMP of PMA/Iono-stimulated human macrophages, slightly protecting them from apoptosis. Human PBMCs-derived macrophages were cultivated for 7 days in the presence of either the Veh., MIM-2, MIM-5, or MIM-10, the medium/treatment being renewed every other day. PMA/Iono and JC-10 probe were co-incubated for 4.5 hours and representative pictures of the fluorescence were taken for each condition. (A) Representative pictures (10X magnification) of the green (520 nm), and the red (570 nm) fluorescence, after treatment of the PBMCs-derived macrophages with the indicated medicines (in one out of the two assessed donors). (B) Green: Red fluorescence ratios were calculated in each of the above-mentioned treatment conditions. The results are presented as the mean ± S.E.M. of the ratios obtained for the two assessed donors. The dotted line highlights the effect of the tested capsules compared with the Veh. condition.