Selenium ameliorates S. aureus -induced inflammation through ROS-mediated NLRP3 inflammasome in bovine mammary epithelial cells

Background Some research has indicated that selenium (Se) plays a significant role during mastitis. However the intracellular anti-inflammatory effect of Se is not fully clear. Due to the ability of Staphylococcus aureus ( S. aureus ) to internalize into host cell, in this study we explored whether Se could regulate inflammation induced by S. aureus through reactive oxygen species (ROS)-mediated NLRP3 inflammasome in bMECs. Result bMECs were treated with 8 μmol/L Na 2 SeO 3 for 12 h before infected with S. aureus for 2 h. Through flow cytometry, Western blot and qPCR analysis, ROS and NLRP3 imflammasome were detected. Result shown that the production of ROS was increased by S. aureus , Se exerted strong inhibitory effects on the production of ROS; The protein expression of NLRP3 inflammasome including NLRP3, ASC and Caspase-1 increased significantly after S. aureus infection, Se played an important role in regulating the expression of NLRP3, ASC and Caspase-1; To further investigate the anti-inflammatory effect of Se, the expression level of IL-1β associated molecule pro-IL-1β and IL-1β were detected. Result shown that the mRNA expression of IL-1β was up-regulated by S. aureus and after Se treatment the expression level of IL-1β mRNA was markedly down-regulated, meanwhile Se play a regulation effect on the protein expression of Pro-IL-1β and IL-1β. Conclusions Here we show that ROS is involved in bMECs inflammation induced by S. aureus and Se ameliorates S. aureus -induced inflammation through ROS-mediated NLRP3 pathway in bMECs.


Abstract
Background Some research has indicated that selenium (Se) plays a significant role during mastitis. However the intracellular anti-inflammatory effect of Se is not fully clear. Due to the ability of Staphylococcus aureus ( S. aureus ) to internalize into host cell, in this study we explored whether Se could regulate inflammation induced by S. aureus through reactive oxygen species (ROS)-mediated NLRP3 inflammasome in bMECs. Result bMECs were treated with 8 μmol/L Na 2 SeO 3 for 12 h before infected with S. aureus for 2 h.
Through flow cytometry, Western blot and qPCR analysis, ROS and NLRP3 imflammasome were detected.
Result shown that the production of ROS was increased by S. aureus , Se exerted strong inhibitory effects on the production of ROS; The protein expression of NLRP3 inflammasome including NLRP3, ASC and Caspase-1 increased significantly after S. aureus infection, Se played an important role in regulating the expression of NLRP3, ASC and Caspase-1; To further investigate the anti-inflammatory effect of Se, the expression level of IL-1β associated molecule pro-IL-1β and IL-1β were detected. Result shown that the mRNA expression of IL-1β was up-regulated by S. aureus and after Se treatment the expression level of IL-1β mRNA was markedly down-regulated, meanwhile Se play a regulation effect on the protein expression of Pro-IL-1β and IL-1β.
Conclusions Here we show that ROS is involved in bMECs inflammation induced by S. aureus and Se ameliorates S. aureus -induced inflammation through ROS-mediated NLRP3 pathway in bMECs.

Background
Mastitis induced by Staphylococcus aureus ( S. aureus) is a serious problem which will causes serious economic losses to the bovine industry [1,2]. Meanwhile due to the ability of S. aureus to internalize into host cell, mastitis induced by S. aureus is often difficult to cure. Along with the intensity and persistence of infection, most animals cannot escape the fate of elimination [3]. Thus finding effective ways to prevent or cure mastitis induced by S. aureus has become research focus on dairy industry.
Innate immunity plays an important role in defensing against pathogens. Once stimulated innate immunity is triggered by pattern recognition receptors (PRRs) [4]. There are two PRRs mainly response for bacterial infection: Toll-like receptors (TLRs) and Nod-like receptors (NLRs) [5,6]. TLRs are located at the cell surface or within endosomal membranes. However NLRs mainly mediate cytosolic recognition of microbial molecules and promote their clearance [7]. Our previous study have already proved that TLRs play a crucial role in regulation inflammation induced by S. aureus in bMECs [8]. However the regulation effect of NLRs on S. aureus intracellular is still not clear, which challenge us for further detailed investigations and understanding.
Unlike TLR2, NLRs were capable to form inflammasomes in response to their specific stimulators [9,10]. Among them the most characterized member of the NLR family is the NLRP3 inflammasome [11]. Many studies have provided the activation process of NLRP3 pathway. Briefly, NlRP3 inflammasome composed of NLRP3, ASC and caspase-1, the activation of NLRP3 inflammasome ultimately process pro-IL-1β into IL-1β. Moreover, induce inflammatory response [12,13]. Originally NLRP3 was hypothesized to be a cytosolic receptor. However with the deepening study, it seems highly improbable that NLRP3 acts as a receptor to directly bind to bacterial stimuli. ROS, produced by many known activators of NLRP3 inflammasomes, are shown to be a critical mechanism triggering NLRP3 inflammasome formation [14]. Furthermore S. aureus have been found to activate the NLRP3 inflammasome in monocytes/macrophages leading to inflammation [15]. However whether ROS offers a linked to the S. aureus infection and the antivation of 4 NLRP3 inflammasome in bMECs is still unknown.
Se, an essential micronutrient, is a widely used feedstuff additive in dairy industry for a series of biology function such as antioxidant and immune anti-inflammation [16,17]. Some research has indicated that Se deficiency is associated with an increased incidence of placental retentions, metritis, mastitis, susceptibility to infections and reduced fertility [18]. On dairy industry, studies demonstrated adding Se in dietary could affect the innate and the adaptive immune responses to mastitis [19]. The underlying mechanisms of Se in regulating mastitis is complicated. Our previous studies have confirmed that Se could ameliorates S. aureus-induced inflammation in bovine mammary epithelial cells by inhibiting activation of TLR2 signaling pathways. However further research is needed to determine whether NLRP3 pathway is involved in mastitis induced by S. aureus and whether Se is involved in the regulation of NLRP3 pathway in bMECs.

Change in ROS level
The result are shown in Fig. 1. Compared with CG (bMECs without any treatment, bMECs, Fig. 1A) and SeG (bMECs treated with Se for 12 h, bMECs + DHR123 + Se, Fig. 1C), the production level of ROS increased significantly in MG (bMECS infected with S. aureus for 2 h, bMECs + DHR123 + S. aureus, Fig. 1D); Compared with MG the production level of ROS was markedly decreased in TG (bMECs treated with Se for 12 h before infected with S. aureus for 2 h, bMECs + DHR123 + Se + S. aureus, Fig. 1E).

Change in NLRP3 inflammasome
As shown in Fig. 2. Compared with CG and SeG the expression of NLRP3 was significantly increased in MG and compared with MG the NLRP3 expression level decreased significantly in TG ( Fig. 2A and B); S. aureus infection markedly increased the expression of ASC and Se treatment significantly decreased the ASC protein expression ( Fig. 2A

Changes in the expression of IL-1β
Result shown that the IL-1β mRNA expression increased significantly in MG, and compared with MG, the expression of IL-1β mRNA was decreased significantly in TG (Fig. 3C); Compared with CG and SeG the protein expression level of Pro-IL-1β was up-regulated by S. aureus in MG, Se exerted strong inhibitory effects on the expression of Pro-IL-1β ( Fig. 3A and B); Compared with CG and SeG, the production of IL-1β increased significantly induced by S. aureus and this effect was blocked by Se in TG ( Fig. 3A and D).

Discussion
Oxidative Stress (OS) is caused by an imbalance between free radical formation and antioxidant defense [20]. And OS induces production of ROS in cells [21]. Due to mastitis itself presents an OS, in this study we first detected the ROS level to understand the impact of S. aureus on bMECs OS. Result shown that the bMECs ROS level increased significantly after S. aureus infection, and Se obviously suppressed the production of ROS induced by S.aureus. The result demonstrated that ROS is involved in bMECs inflammation induced by S. aureus and also Se is involved in regulation the production of ROS in bMECs.
The activation of NLRP3 inflammasomes has been associate with a serious of pathological conditions including mastitis [22,23]. NLRP3 inflammasomes is a tripartite protein that consists of NLRP3, ASC and caspase-1. The NLRP3 protein interacts with ASC to initiate inflammasome assembly [24]. And caspase-1 is mainly responsible for the maturation of inactive cytokine precursors such as IL-1β, a powerful inflammatory mediator, which is one of the most studied cytokines related to the innate immune response [25]. NLRP3 inflammasome activation in response to a several of signals, such as virus, S. aureus and Escherichia coli [ 26,27]. It seems that a broad range of stimuli have been demonstrated to cause NLRP3 activation, however research shown that ROS serves as a triggering factor to activate NLRP3 inflammasomes [28]. Flow cytometry analysis showed that Se play a regulation effect on the production of ROS induced by S. aureus. Therefore we detected the effect of Se on NLRP3 pathway subsequently. Result shown that Se suppressed the expression of NLRP3, ASC and caspase-1, which indicating an inhibitory effect of NLRP3 inflammasome; meanwhile the expression of pro-IL-1β and IL-1β were also suppressed, which indicating that Se could protect bMECs from S. aureus injury.
In conclusion, this study proved the protective effect of Se on S. aureus-induced inflammation in bMECs. This effect was at least partly achieved by the blocking ROS-

Funding
The investigation was supported by the National Natural Science Foundation of China (NO.

Availability of date and materials
All data used during the study appear in the submitted article.

Authors' contributions
Bi CL contributed to the overall study design and supervised all research. Li H analyzed the data and Zhang SJ prepared Figures and contributed partly to writing and finally revising the manuscript and data analysis. Tang H drafted and revised the first version of the manuscript. All the authors reviewed and finally approved the manuscript.

Ethical approval
The protocol was approved by the Animal Care and Ethics Committee of Linyi University.

Competing interests
The authors declare that they have no conflict of interest.