*3.4. Immunoregulatory Effect of EGb Treatment on Cytokine Production by PBLs*

In the light of obtained results of beneficial effect of EGb on the PBLs resistance to viral infection/level of innate immunity, it became interesting to investigate the impact of the extract on cytokine production, which is engaged in innate immune response, as changes in viral replication are related to changes in cytokine balance produced by whole PBLs. Table 5 shows an average level of cytokine production, namely TNF-α, IFN-γ, IL-10, IL-1β, and IL-15, by uninfected (spontaneous release) and VSV-infected PBLs from AD patients and controls. The effect of EGb treatment is also presented. As shown in Table 5, VSV infection of PBLs resulted in high TNF-α but slight IL-1β and IL-15 production in both groups. PBLs infection with VSV also revealed increased IFN-γ release but only in the control group. In the case of IL-10, VSV infection resulted in decreased production of this cytokine in AD patients and controls. Interestingly, EGb treatment differentially influenced on all investigated cytokines.

**Table 5.** Uninfected (spontaneous) and VSV-infected cytokine production by PBLs. Effect of EGb treatment. Median is measure of average level. For every individual person and for every change in cytokine level after EGb, treatment equals d = log *a f ter bef ore* = log(*a f ter*) − log(*bef ore*). EGb effect is average of all *d s* in a group. EGb effect is than average of ratios *a f ter bef ore* expressed in logs. Delta Δ is measure of difference in EGb effects between AD and controls, and this statistic is the average difference between a randomly selected person from AD group and a randomly selected person from control group.



**Table 5.** *Cont.*

EGb decreased **TNF-**α production by uninfected (spontaneous) and VSV-infected PBLs from AD patients and controls. Notably, this effect was considerably higher in AD patients. The average level of spontaneous TNF-α production by PBLs from AD patients was *MedPBLs*; *bef ore TNF*−*α*;*AD* <sup>=</sup> 27.17 pg/mL, and after EGb treatment, it decreased to *MedPBLs*; *a f ter TNF*−*α*;*AD* <sup>=</sup> 1.01 pg/mL. This change (effect) was statistically significant at *<sup>α</sup>* <sup>=</sup> 0.05. In the control group, the average level of spontaneous TNF-α production by PBLs before EGb treatment was *MedPBLs*; *bef ore TNF*−*α*;*Control* <sup>=</sup> 49.02 pg/mL, and after EGb treatment, it decreased to *MedPBLs*; *a f ter TNF*−*α*;*Control* <sup>=</sup> 17.2 pg/mL. This change (effect) was also statistically significant. There was significant difference in this effect between AD patients and controls. The decrease of spontaneous TNF-α production by PBLs from AD patients after EGb treatment was higher than in the control group (*p* = 0.0107). Similarly, the average level of TNF-α produced by VSV-infected PBLs from AD was *MedPBLs*+*VSV*; *bef ore TNF*−*α*;*AD* <sup>=</sup> 60.47 pg/mL, and after EGb treatment, it decreased to *MedPBLs*+*VSV*; *a f ter TNF*−*α*;*AD* <sup>=</sup> 1.1 pg/mL. This change (effect) was statistically significant at *α* = 0.05. For control group, the average level of TNF-α produced by VSV-infected PBLs was *MedPBLs*+*VSV*; *bef ore TNF*−*α*;*Control* <sup>=</sup> 212.83 pg/mL, and after EGb treatment, it decreased to *MedPBLs*+*VSV*; *a f ter TNF*−*α*;*Control* <sup>=</sup> 22.56 pg/mL. This change (effect) was also statistically significant at *α* = 0.05. There was significant difference in this effect between AD patients and controls. The decrease of TNF-α produced by VSV-infected PBLs from AD patients after EGb treatment was higher than in the control group (*p* = 0.0317).

In the case of**IFN-**γ, we can thereby see that EGb decreased spontaneous production of this cytokine in AD group from *MedPBLs*; *bef ore INF*−*γ*−*α*;*AD* <sup>=</sup> 9.84 pg/mL to *MedPBLs*; *af ter IFN*−*γ*−*α*;*AD* <sup>=</sup> 2.69 pg/mL. The change (effect) was statistically significant at *α* = 0.05. This effect was not observed in the control group probably due to a small sample size. Thus, there was significant difference in

EGb effect between AD patients and controls.The decrease of spontaneous INF-γ production in AD patients after EGb treatment was higher than in the control group (*p* = 0.0003). Similarly, after EGb treatment, the decrease in IFN-γ production by VSV-infected PBLs from AD patients was observed—from *MedPBLs*+*VSV*; *bef ore IFN*−*γ*−*α*;*AD* <sup>=</sup> 9.86 pg/mL to *MedPBLs*+*VSV*; *af ter IFN*−*γ*−*α*;*AD* <sup>=</sup> 2.83 pg/mL. The change (effect) was statistically significant at *α* = 0.05. For control group, the average level of INF-<sup>γ</sup> produced by VSV-infected PBLs was *MedPBLs*+*VSV*; *bef ore IFN*−*γ*−*α*;*Control* <sup>=</sup> 8.66 pg/mL and after EGb treatment, it decreased to *MedPBLs*+*VSV*; *af ter IFN*−*γ*−*α*;*Control* <sup>=</sup> 5.05 pg/mL. The change (effect) was also statistically significant at *α* = 0.05. Significant difference in EGb effect between AD patients and controls was noticed. A decrease of INF-γ production by VSV-infected PBLs in AD patients after EGb treatment was higher than in the control group (*p* = 0.0127).

The reducing effect of EGb treatment was shown either for anti-inflammatory cytokine **IL-10** production. EGb decreased spontaneous IL-10 production in AD group from *MedPBLs*; *bef ore IL*−10−*α*;*AD* <sup>=</sup> 86.13 pg/mL to *MedPBLs*; *a f ter IL*−10−*α*;*AD* <sup>=</sup> 10.47 pg/mL. This change was statistically significant. In the control group, EGb decreased IL-10 production from *MedPBLs*; *bef ore IL*−10−*α*;*AD* <sup>=</sup> 162.87 pg/mL to *MedPBLs*; *a f ter IL*−10−*α*;*AD* <sup>=</sup> 28.76 pg/mL. This change was also statistically significant. In the case of IL-10 production after VSV-infection, EGb decreased the level of this cytokine in AD patients from *MedPBLs*+*VSV*; *bef ore IL*−10−*α*;*Control* <sup>=</sup> 43.49 pg/mL to *MedPBLs*+*VSV*; *a f ter IL*−10−*α*;*Control* <sup>=</sup> 2.37 pg/mL. This change was statistically significant. In the control group, EGb also decreased IL-10 production from *MedPBLs*+*VSV*; *bef ore IL*−10−*α*;*Control* <sup>=</sup> 163.73 pg/mL to *MedPBLs*+*VSV*; *a f ter IL*−10−*α*;*Control* <sup>=</sup> 20.11 pg/mL. This change was also statistically significant. A decrease of IL-10 by uninfected and VSV-infected PBLs after EGb treatment was the same in AD patients and controls.

In contrast, EGb increased IL-1β and IL-15 production by uninfected and VSV-infected PBLs from AD and controls. EGb increased **IL-1**β production by uninfected PBLs from control group. The average level of spontaneous IL-1β production was *MedPBLs*; *bef ore IL*1−*β*−*α*;*AD* <sup>=</sup> 288.6 pg/mL, and after EGb treatment, it was *MedPBLs*; *af ter IL*−1*β*−*α*;*AD* <sup>=</sup> 496.12 pg/mL. The change (effect) was statistically significant at *α* = 0.05. This effect was not observed in AD group probably due to a small sample size. Similarly, the average level of IL-1β produced by VSV-infected PBLs from AD was *MedPBLs*+*VSV*; *bef ore IL*−1*β*−*α*;*AD* <sup>=</sup> 401.33 pg/mL, and after EGb treatment, it increased to *MedPBLs*+*VSV*; *af ter IL*−1*β*−*α*;*AD* <sup>=</sup> 427.88 pg/mL. For control group, the average level of IL-1<sup>β</sup> produced by VSV-infected PBLs was *MedPBLs*+*VSV*; *bef ore IL*−1*β*−*α*;*Control* <sup>=</sup> 550.54 pg/mL and after EGb treatment, it increased to *MedPBLs*+*VSV*; *af ter IL*−1*β*−*α*;*Control* <sup>=</sup> 604.51 pg/mL. An increase of IL-1<sup>β</sup> by VSV-infected PBLs after EGb treatment was the same in AD patients and controls.

EGb treatment influenced positively and statistically significant on **IL-15** production by uninfected PBLs from AD and controls. An increase of this cytokine was observed, respectively, in AD from *MedPBLs*; *bef ore IL*−15−*α*;*AD* <sup>=</sup> 22.7 pg/mL to *MedPBLs*; *af ter IL*−15−*α*;*AD* <sup>=</sup> 39.59 pg/mL and in controls from *MedPBLs*; *bef ore IL*−15−*α*;*AD* <sup>=</sup> 30.79 pg/mL to *MedPBLs*; *af ter IL*−15−*α*;*AD* <sup>=</sup> 42.96 pg/mL. In the case of IL-15 production by VSV-infected PBLs, an increased effect of EGb treatment was noticed, respectively, in AD from *MedPBLs*+*VSV*; *bef ore IL*−15−*α*;*Control* <sup>=</sup> 23.7 pg/mL to *MedPBLs*+*VSV*; *af ter IL*−15−*α*;*Control* <sup>=</sup> 39.11 pg/mL and in controls from *MedPBLs*+*VSV*; *bef ore IL*−15−*α*;*Control* <sup>=</sup> 40.71 pg/mL to *MedPBLs*+*VSV*; *af ter IL*−15−*α*;*Control* <sup>=</sup> 48.62 pg/mL. An increase of IL-15 by uninfected and VSV-infected PBLs after EGb treatment was the same in AD patients and controls.

Additionally, the level of **IFN-**α was also investigated. Results showed that PBLs from AD and controls did not produce spontaneous IFN-α, which was expected. In every investigated person in both groups, spontaneous IFN-α production was undetectable. Infection with VSV resulted in high IFN-α production by PBLs in both groups. However, leukocytes from healthy age-matched subjects produced *MedPBLs*+*VSV*; *bef ore IFN*−*α*;*Control* <sup>=</sup> 88.47 pg/mL—over eight times more IFN-<sup>α</sup> than leukocytes from AD patients *MedPBLs*+*VSV*; *bef ore IFN*−*α*;*AD* <sup>=</sup> 10.49 pg/mL. EGb treatment decreased IFN-α, and this effect was very strong, to an undetectable level, in the case of all AD patients and controls. Obtained results were very interesting; however, data were not included in the Table 5 due to experiments that were performed only for half of the participants and need to be confirmed in a larger sample size.

In summary, EGb showed immunoregulatory activity resulted in a different influence on selected pro- and anti-inflammatory cytokine production by PBLs. It decreased TNF-α, IFN-γ, and IL-10 production but increased IL-1β and IL-15 production by uninfected and VSV-infected leukocytes of AD patients and controls. The effects of EGb treatment, however, were much stronger in AD patients. Results of EGb treatment on cytokine production are presented in Figure 3.

**Figure 3.** Influence of EGb on pro- and anti-inflammatory cytokine production by uninfected (spontaneous) and VSV-infected PBLs from AD patients and healthy age-matched controls. EGb effect was measured as natural logarithm of ratio after/before cytokine level. Zero means no effect. Points represent average change after treatment with confidence interval CI95% for the average.
