3.1. D3T-mediated induction of antioxidants in RAW 264.7 cells
We previously showed that D3T induced a host of cellular antioxidant defenses in primary peritoneal macrophages isolated from mice [9]. For the present study, we first determined if D3T also induced antioxidants in RAW 264.7 cells, a mouse macrophage cell line. As shown in Fig. 1, incubation of RAW 264.7 cells with D3T for 24 h resulted in significant induction in cellular GSH, GPx, GR, GST, catalase, and NQO1. The induction of the above antioxidants, except for catalase, showed a D3T-concentration dependency. It remains unclear why catalase induction was independent of D3T concentrations used (25–100 µM). It is possible that 25 µM D3T was enough to cause the maximal induction of catalase under the present experimental conditions.
GSH, GPx, GR, and GST are members of the glutathione system—a major cellular antioxidant defense system involved in the detoxification of various reactive oxygen and nitrogen species (ROS/RNS) [12]. On the other hand, catalase is an enzyme selective for decomposing hydrogen peroxide, whereas NQO1 acts as both a quinone-detoxifying enzyme and a superoxide scavenger [13]. As ROS/RNS are critical players in dysregulated inflammatory responses [14], induction of the above cellular antioxidants would render macrophages more resistant to oxidative stress and dampen macrophage’s proinflammatory responses. Thus, this antioxidant induction experiment would set a stage for the subsequent investigation of the effects of D3T on LPS-induced proinflammatory responses.
3.2. D3T-mediated suppression of LPS-induced proinflammatory responses in RAW 264.7 cells
Release of proinflammatory cytokines by macrophages has been shown to play a critical role in many inflammatory disorders, especially the acute phase of sepsis [13, 15]. We next determined if D3T pretreatment could suppress LPS-induced production of IL-1β and TNF-α, two major proinflammatory cytokines, in macrophages. As shown in Fig. 2, under the basal unstimulated condition, RAW 264.7 cells released minimal amounts of IL-1β and TNF-α; however, exposure of the cells to LPS led to dramatically increased release of both IL-1β and TNF-α into the culture media. Notably, pretreatment of the cells with D3T resulted in a marked concentration-dependent attenuation of LPS-induced IL-1β release. A 71%, 89%, and 94% reduction in LPS-induced IL-1β release was seen with 25, 50, and 100 µM D3T pretreatment, respectively (Fig. 2A). In contrast, the effect of D3T pretreatment on LPS-induced TNF-α release was much less remarkable; only the highest concentration of D3T caused a 28% reduction (Fig. 2B). This differential effect on IL-1β versus TNF-α suggests that different signaling pathways may be involved in the LPS-induced release of these two proinflammatory cytokines. Indeed, a previous study showed that blocking mitochondrial ROS production blunted LPS-induced IL-1β expression without affecting LPS-induced TNF-α expression [8].
To investigate the effect of D3T pretreatment on ROS/RNS production, we measured the total nitric oxide production by RAW 264.7 cells. In line with the effects on the proinflammatory cytokines, LPS exposure dramatically increased the production of total nitric oxide (as assessed by measuring nitrate/nitrite formation) in the cells. The LPS-induced RNS production was significantly blunted by D3T pretreatment in a concentration-dependent manner (Fig. 3). It remains unclear how D3T pretreatment reduced LPS-induced nitric oxide production. It is well established that LPS activates inducible nitric oxide synthase (iNOS), leading to increased production of nitric oxide in macrophages [16, 17]. The suppression by D3T on LPS-induced nitric oxide production in RAW 264.7 cells might result from two potential mechanisms: (i) scavenging nitric oxide by the D3T-induced cellular antioxidants (e.g., GSH) and (ii) suppression of LPS-induced NF-κB activation (see Sect. 3.3). In this context, LPS-induced iNOS expression occurs via an NF-κB-dependent mechanism [18].
3.3. D3T-mediated suppression of LPS-induced NF-κB activation in RAW 264.7 cells
Since NF-κB is a chief regulator of the gene expression of proinflammatory cytokines (including IL-1β and TNF-α) as well as iNOS [19], we determined if D3T pretreatment could also suppress LPS-induced NF-κB activation. As shown in Fig. 4, the LPS-stimulated NF-κB activation was significantly suppressed by D3T pretreatment. However, the degree of NF-κB suppression by D3T was not in line with the extent of D3T-mediated IL-1β suppression (Fig. 2A). In this regard, the suppression of LPS-induced NF-κB activation by D3T did not show a concentration dependency and only a 25–35% inhibition was seen with D3T (25–100 µM) pretreatment (Fig. 4). On the other hand, LPS-induced IL-1β production was suppressed by 71–94% following D3T pretreatment. This discrepancy suggests that inhibition of NF-κB signaling is only one of the mechanisms of D3T-mediated blockage of LPS-induced IL-1β production in macrophages.
3.4. D3T-mediated suppression of LPS-induced proinflammatory responses in Nrf2+/+ and Nrf2−/− peritoneal macrophages
We previously showed that D3T-mediated induction of antioxidants in macrophages was dependent on Nrf2 status [9] and D3T is a potent activator of Nrf2 as well as an inducer of Nrf2 expression [7]. Accordingly, we determined if Nrf2 status could also affect the ability of D3T to suppress LPS-induced proinflammatory cytokine release. To this end, we used peritoneal macrophages isolated from Nrf2-null (Nrf2−/−) mice and the wild-type (Nrf2+/+) mice. As shown in Fig. 5, the suppressing effect of D3T pretreatment on LPS-induced IL-1β and TNF-α release was completely lost in the Nrf2−/− macrophages, suggesting that Nrf2 signaling is indispensable for the anti-proinflammatory activity of D3T in macrophages. Notably, the absence of Nrf2 resulted in a drastic augmentation of the LPS-induced release of TNF-α (Fig. 5B); there was also an increasing trend in LPS-induced IL-1β release, but it did not reach statistical significance due to large sample variations (Fig. 5A). Nevertheless, this finding suggests that the inducible express of proinflammatory cytokines, especially TNF-α, could readily become out of control in the absence of functional Nrf2 signaling, causing overt inflammatory stress. Indeed, targeted disruption of Nrf2 gene has been shown to dramatically sensitize animals to inflammatory tissue injury in an experimental sepsis animal model [7, 20].
In conclusion, the results of the present study demonstrated that D3T—a novel cruciferous dithiolethione-related compound, is effective in attenuating LPS-induced proinflammatory stress in macrophages, and Nrf2-dependent antioxidant induction as well as suppression of NF-κB activation may collectively contribute to the ability of D3T to protect against experimental sepsis observed previously in mice [7].