MATRIZ DE TRIANGULACIÓN DE DATOS Categoría: Competencia “Construye su identidad”

We assessed the effect of co-exposure to AgNPs and a viral infection on IL-6 and CXCL-8 production performing a chequerboard analysis of AgNPs in combination with Poly I:C (Tables 2.7, IL-6 and CXCL-8). In order to calculate the effects of this combination in terms of synergism/antagonism, we analyzed the data using the Chou-Talalay method [167], the most accredited mathematical model developed to calculate drug combination effects. Along with the theory, Chou and Talalay have also elaborated software that calculates the type of effects (synergistic, additive or antagonistic) resulting from a particular drug combination [167]. However, due to the

nature of our data (refer to Section 2.5.3), little statistical reliance could be placed on the results obtained. Therefore, since no documented mathematical model has been applied to the analysis of our data, the terms “synergistic” (potentiation) or

“antagonistic” that we used to describe the effects we measured in the combinations are limited to our experiments and observations. In order to simplify the understanding of our data and of a possible mechanism of action, we generated “heat maps” as previously described for cytotoxicity studies (Sections 2.4.3.4, 2.4.3.5).

TT1 cells were initially exposed to increasing concentrations of AgNPs and, after 1h, to increasing concentrations of Poly I:C or LPS for a further 23h. The combination of AgNPs and Poly I:C resulted in a potentiation (more than the sum of the two) of IL-6 production (Table 2.7, IL-6; red cells), with a few exceptions (yellow cells).

Interestingly, in CXCL-8 release, the type of effect measured seemed to be antagonistic (less than the sum of the two) for every combinations of AgNPs-Poly I:C (Table 2.7, CXCL-8; yellow cells).

IL-6

AgNPs 0 126.47 247.27 1702.61 1993.87 2346.25

AgNPs 1 118.87 255.48 2111.39 2199.48 2200.70

AgNPs 10 203.94 444.91 2176.06 2398.60 2472.72

AgNPs 30 507.89 957.83 2889.68 3415.10 3677.98

AgNPs 50 1122.4 1574.10 3489.37 3397.33 3583.28

Tables 2.7 Effect of co-exposure to AgNPs and Poly I:C on IL-6 and CXCL-8 release. TT1 cells were exposed first to increasing concentrations of AgNPs (μg/ml) and, after 1h, to increasing concentrations of Poly I:C (µg/ml) for another 23h. IL-6 and CXCL-8 expression over 24h was evaluated by ELISA. Data are presented as the mean of three independent experiments; (n=3).

 antagonistic effect;  additive/synergistic effect

In order to better visualise the differences between IL-6 and CXCL-8 release from co-exposed TT1 cells, values from the heat map were plotted into a graph, choosing only one concentration of Poly I:C against all the concentrations of AgNPs (Figure 2.14).

Showing insignificant decrease in cell viability and sub-optimal release of IL-6 and CXCL-8, 20µg/ml was established as the concentration of Poly I:C for subsequent experiments. Thus, we plotted the values for Poly I:C 20µg/ml (pink bars) combined with all the increasing concentrations of AgNPs (light blue bars) into a graph. At every concentration of AgNPs, the combination with 20μg/ml of Poly I:C resulted in a potentiation of IL-6 production. In fact, the combined effect (dark blue bars) was higher

CXCL-8

AgNPs 0 436.32 599.93 699.68 1188.38 1334.42

AgNPs 1 341.09 451.32 652.46 812.56 797.37

AgNPs 10 600.15 773.66 1147.17 1039.98 953.10

AgNPs 30 1093.8 1038.47 1337.10 1256.33 1461.64

AgNPs 50 1515.6 1218.98 1755.60 1523.84 1814.89

than the mathematical sum (horizontal orange lines) of the same effect. For every combination of AgNP and Poly I:C, we calculated two different values: the mathematical sum of the two single effects (horizontal orange lines) and the actual combined effect (dark blue lines). For example, for the combination AgNPs 50μg/ml and Poly I:C 20μg/ml (Table 2.7, heat map for IL-6), the mathematical sum (expressed in pg/ml) was 2825.01pg/ml (1122.40+1702.61), but the actual combined effect was 3489.37. Therefore in this case, being the combined effect (3489.37) higher than the mathematical sum (2825.01pg/ml), the co-exposure seemed to result in a potentiation of the two single effects. The same applied to the other combinations.

On the contrary, every combination of AgNPs and Poly I:C seemed to suggest an antagonistic effect on CXCL-8 production (the combined effect was lower than the mathematical sum; Figure 2.14).

IL-6

NT 1 10 30 50

0 1000 2000 3000 4000

Ag g/ml Poly 20g/ml

Ag+Poly (combined effect) Ag+Poly (mathematical sum) [Ag] (g/ml)

IL-6 (pg/ml)

CXCL-8

NT 1 10 30 50

0 1000 2000 3000

[Ag] (g/ml)

CXCL-8 (pg/ml)

Figure 2.14 Effect of co-exposure to AgNPs and Poly I:C on IL-6 and CXCL-8 release. TT1 cells were exposed first to increasing concentrations of AgNPs (µg/ml;

light blue bars) and, after 1h, to Poly I:C (20µg/ml; pink bars) for another 23h. IL-6 and 8 expression over 24h was evaluated by ELISA. The amount of IL-6 and CXCL-8 measured at every combination is represented by dark blue bars while the mathematical sum of the 2 single effects (AgNPs + Poly I:C) is indicated by the orange lines. NT=non treated. Data are presented as the mean ± SEM; (n=3).

2.4.3.7.5 Effect of co-exposure to AgNPs and LPS on IL-6 and CXCL-8 release

In order to investigate the effect of AgNP exposure followed by a bacterial infection, the same analysis described for Poly I:C was repeated using LPS. In contrast to Poly I:C, AgNPs-LPS combination did not result in a synergism of IL-6, except for two combinations with AgNPs 50µg/ml (Table 2.8 IL-6, red cells). Conversely, the heat map generated for CXCL-8 release suggested that some of the combinations of AgNPs (30 and 50µg/ml) with LPS have a synergistic effect (Table 2.8 CXCL-8, red cells).

IL-6

AgNPs 0 7.38 10.09 15.67 21.77 34.73 154.81

AgNPs 1 13.04 14.59

AgNPs 0 142.39 201.32 184.04 227.78 235.62 159.20

AgNPs 1 162.17 207.56

Tables 2.8 Effect of co-exposure to AgNPs and LPS on IL-6 and CXCL-8 release.

TT1 cells were exposed first to increasing concentrations of AgNPs (μg/ml) and, after 1h, to increasing concentrations of LPS (ng/ml) for another 23h. IL-6 and CXCL-8 expression over 24h was evaluated by ELISA. The values in brackets are the result of every measured value against its sum total. Data are presented as the mean of three independent experiments; (n=3).

 antagonistic effect;  additive/synergistic effect.

Showing an insignificant decrease in cell viability and sub-optimal release of IL-6, 1000ng/ml was established as the concentration of LPS for subsequent experiments.

As for Poly I:C, we plotted the values (from the heat map) corresponding to 1000ng/ml LPS against all the concentrations of AgNPs in a bar graph (Figure 2.15). The addition of LPS (green bars) to every concentration of AgNPs (light blue bars) resulted in a combined effect (dark blue bars) lower than the corresponding mathematical sum (orange lines), for both IL-6 and CXCL-8 release. This suggested an antagonistic effect.

IL-6

NT 1 10 30 50

0 25 50 2000 4000

Ag g/ml LPS 1000ng/ml

Ag+LPS (combined effect) Ag+LPS (mathematical sum) [Ag] (g/ml)

IL-6 (pg/ml)

CXCL-8

NT 1 10 30 50

0 1500 3000 4500

[Ag] (g/ml)

CXCL-8 (pg/ml)

Figure 2.15 Effect of co-exposure to AgNPs and LPS on IL-6 and CXCL-8 release.

TT1 cells were exposed first to increasing concentrations of AgNPs (µg/ml; light blue bars) and, after 1h, to LPS (1000ng/ml; green bars) for another 23h. IL-6 and CXCL-8 expression over 24h was evaluated by ELISA. The amount of IL-6 and CXCL-8 measured at every combination is represented by dark blue bars while the mathematical sum of the 2 single effects (AgNPs + LPS) is indicated by the orange lines. NT=non treated. Data are presented as the mean ± SEM; (n=3).

2.4.4 Effect of AgNPs on MAP-kinase pathway