Immunomodulatory Effects of the Tobacco Defensin NaD1

Abstract

Background/Objectives: Defensins are important components of the innate plant immune system, exhibiting antimicrobial activity against phytopathogens, as well as against fungi pathogenic to humans. Along with antifungal activity, plant defensins are also capable of influencing various immune processes, but not much is known about these effects. In this study, we investigated the immunomodulatory effects of the tobacco defensin NaD1, which possesses a pronounced antifungal activity. Methods and Results: We showed that NaD1 could penetrate the Caco-2 polarized monolayer. Using a multiplex assay with a panel of 48 cytokines, chemokines and growth factors, we demonstrated that NaD1 at a concentration of 2 μM had immunomodulatory effects on human dendritic cells and blood monocytes, mainly inhibiting the production of various immune factors. Using the sandwich ELISA method, we demonstrated that NaD1 at the same concentration had a pronounced immunomodulatory effect on unstimulated THP-1-derived macrophages and those stimulated by bacterial LPS or fungal zymosan. NaD1 had a dual effect and induced the production of both pro-inflammatory cytokine IL-1β as well as anti-inflammatory IL-10 on resting and pro-inflammatory THP-1-derived macrophages. We also found that the immunomodulatory effects of the tobacco defensin NaD1 and the pea defensin Psd1 differed from each other, indicating nonuniformity in the modes of action of plant defensins. Conclusions: Thus, our data demonstrated that the tobacco defensin NaD1 exhibits different immunomodulatory effects on various immune cells. We hypothesized that influence on human immune system along with antifungal activity, could determine the effectiveness of this peptide under infection in vivo.

Keywords: antimicrobial peptides (AMPs); cytokines; host defense peptides; human cathelicidin LL-37; human β-defensin 2 (HBD2); immunomodulatory effects; lipopolysaccharide (LPS); pea defensin Psd1; plant defensins; tobacco defensin NaD1; zymosan.

Figure 1

Cytotoxic effects of the tobacco defensin NaD1 towards PBMCs (A) and Caco-2 cells in monolayer (C). The membrane-active peptide melittin from the venom of honeybees (B,D) was used for comparison. Error bars represent a standard deviation (±SD) between two biological and two technical replications. Significance levels are * p ≤ 0.05, *** p < 0.001 and **** p < 0.0001. The significance was calculated by comparing untreated cells (control) with treated by NaD1 or melittin cells. Viability cells in control and experimental samples was compared with un-paired two-sample t-test.

Figure 2

Assessment of bidirectional transport of the tobacco defensin NaD1 through the polarized Caco-2 monolayer. A→B, absorptive transport; B→A, secretory transport; Papp—apparent permeability coefficient. Six and four independent biological replications were used for absorptive and secretory directions, respectively. The normality of Papp coefficient distribution was assessed using Shapiro–Wilk test. Papp coefficients were compared by unpaired two-sample t-test.

Figure 3

Production of cytokines, chemokines and growth factors upon stimulation of DCs and monocytes by NaD1 at the concentration of 2 μM. Error bars represent a standard deviation (±SD) between two biological replications. The levels in control and experimental wells were compared by unpaired two-sample t-test.

Figure 4

Influence of the tobacco defensin NaD1 and other AMPs at the concentration of 2 µM on production of pro- (A–D) and anti-inflammatory (B) cytokines either unstimulated or stimulated by LPS or by zymosan THP-1-derived macrophages. Error bars represent a standard deviation (±SD) between two biological and two technical replications. Significance levels are * p ≤ 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. The significance of difference in cytokine production was calculated by comparing: unstimulated cells (control) with stimulated by AMPs cells (grey bars); stimulated by LPS (blue bars) or zymosan (green bars) cells alone or in the presence of AMPs. Release of the cytokines in control and experimental samples was compared with unpaired two-sample t-test.