Personalised Profiling of Innate Immune Memory Induced by Nano-Imaging Particles in Human Monocytes

Abstract

Engineered nanoparticles used for medical purposes must meet stringent safety criteria, which include immunosafety, i.e., the inability to activate possibly detrimental immune/inflammatory effects. Even medical nanomaterials devoid of direct immunotoxic or inflammatory effects may have an impact on human health if able to modify innate memory, which is the ability to "prime" future immune responses towards a different, possibly more detrimental reactivity. Although innate memory is usually protective, anomalous innate memory responses may be at the basis of immune pathologies. In this study, we have examined the ability of two nanomaterials commonly used for diagnostic imaging purposes, gold and iron oxide nanoparticles, to induce or modulate innate memory, using an in vitro model based on human primary monocytes. Monocytes were exposed in culture to nanoparticles alone or together with the bacterial agent LPS (priming phase/primary response), then rested for six days (extinction phase), and eventually challenged with LPS (memory/secondary response). The memory response to the LPS challenge was measured as changes in the production of inflammatory (TNFα, IL-6) and anti-inflammatory cytokines (IL-10, IL-1Ra), as compared to unprimed monocytes. The results show that both types of nanoparticles can have an effect in the induction of memory, with changes observed in the cytokine production. By comparing nanomaterials of different shapes (spherical vs. rod-shaped gold particles) and different size (17 vs. 22 nm diameter spherical iron oxide particles), it was evident that innate memory could be differentially induced and modulated depending on size, shape and chemical composition. However, the main finding was that the innate memory effect of the particles was strongly donor-dependent, with monocytes from each donor showing a distinct memory profile upon priming with the same particles, thereby making impossible to draw general conclusions on the particle effects. Thus, in order to predict the effect of imaging nanoparticles on the innate memory of patients, a personalised profiling would be required, able to take in consideration the peculiarities of the individual innate immune reactivity.

Keywords: imaging materials; innate immunity; innate memory; monocytes; nanoparticles.

Figure 1

Size and morphology of the AuNP and FeOxNP used in this study. TEM images of spherical AuNP (upper left panel), rod-shaped AuNP (upper right panel), FeOxNP 17 (lower left panel) and FeOxNP 22 (lower right panel). Size bars (50 nm) are shown in each panel.

Figure 2

Innate immune primary response of human monocytes to NP alone or upon co-exposure with LPS. Human monocytes isolated from blood of four individual donors (red, green, blue, and yellow symbols) were cultured for 24 h in culture medium alone (empty columns) or containing serum-precoated NP: 5.7 µg/ml spherical AuNP (AuNP SPH), 1.4 µg/ml rod-shaped AuNP (AuNP ROD), 2 µg/ml spherical FeOxNP of 16.8 nm diameter (FeOxNP 17) and 2.7 µg/ml spherical FeOxNP of 22.4 nm diameter (FeOxNP 22) alone (light blue columns) or together with 1 ng/ml LPS (orange columns). The production of TNFα (upper left), IL-6 (upper right), IL-10 (lower left) and IL-1Ra (lower right) was measured in the 24 h supernatants by ELISA. Data are presented as individual donors’ values (coloured symbols) ± SD and as mean of the individual values (dotted columns).

Figure 3

Innate immune memory response of human monocytes primed with NP alone or together with LPS. Human monocytes isolated from blood of four individual donors (red, green, blue, and yellow symbols) were cultured for 24 h in culture medium alone or containing serum-precoated NP: 5.7 µg/ml AuNP SPH, 1.4 µg/ml AuNP ROD, 2 µg/ml FeOxNP 17 and 2.7 µg/ml FeOxNP 22 alone (light blue columns) or together with 1 ng/ml LPS (orange columns) (line priming in abscissa). Cells were then washed and rested for 6 days in the absence of stimuli, then challenged for 24 h in fresh medium alone or containing 5 ng/mL LPS (m and LPS in the abscissa line challenge). The production of TNFα (upper left), IL-6 (upper right), IL-10 (lower left) and IL-1Ra (lower right) was measured in the 24 h supernatants by ELISA. Data are presented as individual donors’ values (coloured symbols) ± SD and as mean of the individual values (dotted columns). The basal response of control cells (CTR), i.e., primed or unprimed cells rested for 6 days and then exposed for 24 h to medium alone, is shown at the extreme left in each panel (empty columns).

Figure 4

Innate memory response of human monocytes from individual donors, primed with AuNP or FeOxNP alone or together with LPS. Human monocytes isolated from blood of two individual donors (donor 3, square symbols, left panels; donor 2, triangle symbols, right panels) were cultured for 24 h in culture medium alone or containing serum-precoated NP: 5.7 µg/ml AuNP SPH, 1.4 µg/ml AuNP ROD, 2 µg/ml FeOxNP 17 and 2.7 µg/ml FeOxNP 22, alone or together with 1 ng/ml LPS (line priming in abscissa). Cells were then washed and rested for 6 days in the absence of stimuli, then challenged for 24 h in fresh medium alone or containing 5 ng/mL LPS (m and LPS in the abscissa line challenge). The production of TNFα (upper left), IL-6 (upper right and lower left), and IL-1Ra (lower right) was measured in the 24 h supernatants by ELISA. Data are reported as mean ± SD of replicate determination from individual donors out of four tested (all shown in Figure 3). Horizontal dotted lines represent the reference values of cells primed with medium alone and cells primed with LPS in the two parts of each panel. The basal response of control cells (CTR), i.e., primed or unprimed cells rested for 6 days and then exposed for 24 h to medium alone, is shown at the extreme left in each panel (grey symbols). Statistical significance is as follows. Upper left panel: CTR vs. medium, p <0.0001; medium vs. LPS, p <0.005; medium vs. FeOxNP, p <0.005; LPS vs. LPS + AuNP, p <0.05. Upper right: CTR vs. medium, p <0.0001; medium vs. LPS, p <0.005; medium vs. AuNP SPH and FeOxNP 22, p <0.05; LPS vs. LPS + FeOxNP 17, p <0.05. Lower left: CTR vs. medium, p <0.0001; medium vs. LPS, p <0.01; medium vs. AuNP ROD, FeOxNP 17 and FeOxNP 22, p <0.05; LPS vs. LPS + FeOxNP 17, p <0.05. Lower right: medium vs. FeOxNP 17, p <0.05; LPS vs. LPS + all NP, p <0.05. All other relevant comparisons are not significant.

Figure 5

Anti-inflammatory innate memory response of human monocytes from one individual donor, primed with FeOxNP alone or together with LPS. Human monocytes isolated from blood of one individual donor (donor 4, diamond symbols); were cultured for 24 h in culture medium alone or containing serum-precoated FeOxNP (2 µg/ml FeOxNP 17 and 2.7 µg/ml FeOxNP 22) alone or together with 1 ng/ml LPS (line priming in abscissa). Cells were then washed and rested for 6 days in the absence of stimuli, then challenged for 24 h in fresh medium alone or containing 5 ng/mL LPS (m and LPS in the abscissa line challenge). The production of IL-10 (upper panel) and IL-1Ra (lower panel) was measured in the 24 h supernatants by ELISA (complete data shown in Figure 3). Horizontal dotted lines represent the reference values of cells primed with medium alone and cells primed with LPS in the two parts of each panel. The basal response of control cells (CTR), i.e., primed or unprimed cells rested for 6 days and then exposed for 24 h to medium alone, is shown at the extreme left in each panel (grey symbols). Data are reported as mean ± SD of replicate determination from individual donors out of four tested (all shown in Figure 3). Statistical significance is as follows. Upper panel: medium vs. LPS, p <0.05; medium vs. both FeOxNP, p <0.05; LPS vs. LPS + FeOxNP, p <0.05; FeOxNP 17 vs. LPS + FeOxNP 17, p <0.05. Lower panel: medium vs. FeOxNP 17, p <0.05; FeOxNP 17 vs. FeOxNP22, p <0.05. All other relevant comparisons are not significant.