How to Address the Adjuvant Effects of Nanoparticles on the Immune System

Abstract

As the nanotechnology market expands and the prevalence of allergic diseases keeps increasing, the knowledge gap on the capacity of nanomaterials to cause or exacerbate allergic outcomes needs more than ever to be filled. Engineered nanoparticles (NP) could have an adjuvant effect on the immune system as previously demonstrated for particulate air pollution. This effect would be the consequence of the recognition of NP as immune danger signals by dendritic cells (DCs). The aim of this work was to set up an in vitro method to functionally assess this effect using amorphous silica NP as a prototype. Most studies in this field are restricted to the evaluation of DCs maturation, generally of murine origin, through a limited phenotypic analysis. As it is essential to also consider the functional consequences of NP-induced DC altered phenotype on T-cells biology, we developed an allogeneic co-culture model of human monocyte-derived DCs (MoDCs) and CD4+ T-cells. We demonstrated that DC: T-cell ratios were a critical parameter to correctly measure the influence of NP danger signals through allogeneic co-culture. Moreover, to better visualize the effect of NP while minimizing the basal proliferation inherent to the model, we recommend testing three different ratios, preferably after five days of co-culture.

Keywords: DC:T-cell co-culture; MoDCs; amorphous silica nanoparticles; danger signal; in vitro models and methods; maturation; monocyte-derived dendritic cells; nanotoxicology.

Figure 1

Allogeneic model of DCs and CD4 + T-cells co-culture with the different ratios tested. Cells are incubated in presence or absence of 12.5 μg·mL⁻¹ of aSNP for 16 h. Treated MoDCs are co-cultured with allogeneic CD4+ T-cells labeled with CFSE at a ratio of 1:20 (1 MoDC for 20 CD4+ T-cells), 1:50 and 1:100). Proliferation is quantified after 5 or 6 days of co-culture as the percentage of CFSE^low CD4+ T-cells.

Figure 2

Typical TEM images of unstained fumed silica nanoparticles suspended in water. Aqueous suspension of nanoparticles was deposited onto a formvar/carbon coated copper grid and observed using a JEOL 1400 TEM instrument operating at 120 kV. Analysis of recorded images was performed using ImageJ 1.52 software.

Figure 3

Nanoparticles induced HLA-DR expression. Cells were incubated in the presence or absence of LPS as a positive control, or 12.5 and 25 μg·mL⁻¹ of aSNP for 16 h. Cells were then collected, washed and the surface expression of HLA-DR was assessed by FACS analysis. Untreated DCs were used as negative control. Results are expressed as percentage of positive cells (a) or relative fluorescence intensities (b) and represented the mean ± SEM of three independent experiments. Tukey’s honest significance test was employed in conjunction with One-Way ANOVA **: p < 0.01.

Figure 4

Lymphocyte proliferation reached a plateau at high ratios after 6 days of co-culture. Cells were incubated in the presence or absence of 12.5 μg·mL⁻¹ of aSNP for 16 h. Treated MoDCs were co-cultured with allogeneic CD4+ T-cells labeled with CFSE at the ratios of 1:5 (1 MoDC per 5 CD4+ T-cells), 1:10 and 1:20). Proliferation was quantified after 6 days of co-culture as the percentage of CFSE^low CD4+ T-cells. Untreated DCs were used as negative control. Results are shown as percentage of lymphocyte proliferation using FACS histogram (a) or curve (b) for a representative experiment.

Figure 5

Low ratios allowed a better visualization of the increase in lymphocyte proliferation after 6 days of co-culture. Cells were incubated in the presence or absence of 12.5 μg·mL⁻¹ of aSNP for 16 h. Treated MoDCs were co-cultured with allogeneic CD4+ T-cells labeled with CFSE at a ratio of 1:20 (1 moDC for 20 CD4+ T-cells), 1 :50 and 1 :100). Proliferation was quantified after 6 days of co-culture as the percentage of CFSE^low CD4+ T-cells. Untreated DCs were used as negative control. Results are shown as percentage of lymphocytes proliferation using FACS histogram (a) or curve (b) for a representative experiment.

Figure 6

aSNP induced an increase in lymphocyte proliferation after 5 days of co-culture. Cells were incubated in the presence or absence of 12.5 μg·mL⁻¹ of aSNP for 16 h. Treated MoDCs were co-cultured with allogeneic CD4+ T-cells labeled with CFSE at ratios of 1:20 (1 moDC for 20 CD4+ T-cells), 1:50 and 1:100). Proliferation was quantified after 5 days of co-culture as the percentage of CFSE^low CD4+ T-cells. Untreated DCs were used as negative control. Results are shown as percentage of lymphocyte proliferation using FACS histogram (a) or curve (b) for a representative experiment.

Figure 7

The 5-days incubation period is optimal to highlight lymphocyte proliferation augmentation induced by aSNP. Cells were incubated in the presence or absence of 12.5 μg.mL⁻¹ of aSNP for 16 h. Treated MoDCs were co-cultured with allogeneic CD4+ T-cells labeled with CFSE at ratios of 1:20 (1 moDC for 20 CD4+ T-cells), 1 :50 and 1 :100). Proliferation was quantified after 5 or 6 days of co-culture as the percentage of CFSE^low CD4+ T-cells. Untreated DCs were used as negative control. Results are showed as the Δ (aSNP loaded DC-unloaded DCs), corresponding to the proliferation of NP-treated DCs-proliferation of untreated DCs, and represent the mean ± SEM of three independent experiments. Tukey’s honest significance test was employed in conjunction with Two-way ANOVA *: statistically significant compared to 1:20 ratio with p < 0.05, **** : p < 0.0001.