Walnut Polyphenol Extract Protects against Malathion- and Chlorpyrifos-Induced Immunotoxicity by Modulating TLRx-NOX-ROS

Malathion (MT) and chlorpyrifos (CPF) are immunotoxic organophosphate pesticides that are used extensively in agriculture worldwide. Dietary polyphenols protect against a variety of toxins. In this study, walnut polyphenol extract (WPE) prevents MT- or CPF-induced toxicity to splenic lymphocytes in vitro. WPE promotes the proliferation of MT-exposed splenocytes, as indicated by increases in the proportions of splenic T-lymphocyte subpopulations (CD3+, CD4+, and CD8+ T cells) and levels of T-cell-related cytokines interleukin (IL)-2, interferon-γ, IL-4, and granzyme B, and decreases the apoptosis-associated proteins Bax and p53. WPE also significantly enhances the proliferation of CPF-exposed splenic B lymphocytes (CD19+ B cells) and levels of the B-cell-related cytokine IL-6, leading to decreases of the apoptosis-associated proteins Bax and p53. These effects are related to reduced production of reactive oxygen species (ROS), as evidenced by normalized hydroxyl radical (•OH), malondialdehyde (MDA), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), catalase (CAT), and glutathione (GSH) levels, which are associated with decreased expression of NADPH oxidase 2 (NOX2) and dual oxidase 1 (DUOX1). WPE inhibits the production of ROS and expression of NOX by regulating toll-like receptors 4 and 7 in MT- and CPF-exposed splenic lymphocytes. In conclusion, WPE protects against MT- or CPF-mediated immunotoxicity and inhibits oxidative damage by modulating toll-like receptor (TLR)x-NOX-ROS.


Preparation of Splenocytes
Splenocytes were prepared following the protocols of Yang et al. [27]. Briefly, the mice were euthanized by cervical dislocation and their spleens were removed. Single cell suspensions were prepared in RPMI 1640 (supplemented with 10% fetal bovine serum, 100 U penicillin/mL, 100 mg streptomycin/mL, and 2 mM L-glutamine). In this study, splenocytes stimulated with Con A (5 µg/mL) or LPS (10 µg/mL) were used to investigate the proliferation of splenic T or B cells.

Preparation of the WPE
The WPE was extracted as previously described [27]. Briefly, walnuts (30 g) were stored at -20 • C; the shelled kernels were ground and then immersed in acetate buffer (100 mM, 240 mL), pH 4.8/acetone (30:70, v/v) for 24 h at 4 • C and this process was repeated. The extracts were concentrated using a rotary evaporator until the organic solvent was completely evaporated. The concentrated solution was extracted three times with 75 mL ethyl acetate, and then evaporated to remove the ethyl acetate, and lyophilized; the powder was the WPE.

Flow Cytometry
Lymphocyte phenotypes were analyzed by flow cytometry, as described previously [30]. In short, the splenocytes were washed and diluted to 2.5 × 10 7 cells/mL in PBS. The cells were stained with 1 µg/mL specific FITC-labeled antibody against CD3, CD4, CD8, and CD19, and then stored in the dark at 4 • C for 30 min. After washing with PBS three times, the splenocytes were transferred to fluorescence-activated cell sorting (FACS) tubes to measure the CD3+, CD4+, and CD8+ T cells and CD19+ B cell subset levels by lymphocyte phenotype analysis. The results were determined Nutrients 2020, 12, 616 4 of 20 using a Becton Dickinson FACSCalibur flow cytometer (San Diego, CA, USA). The results were calculated as the percentage of positive cells recorded by the flow cytometer within a gate that both exposed and controlled splenocytes. All data were analyzed with Emerald Biotech FlowJo software (Hangzhou, China).

ELISA
Splenocytes were incubated with the test reagents (at a density of 5 × 10 6 cells/mL medium) for 48 h in 96-well plates. The IL-2, IL-4, IL-6, IFN-γ, and granzyme-B levels were measured using commercial ELISA kits.

Antioxidant Enzyme Activities and Biomarkers of Oxidative Stress
To assess the antioxidant enzyme activities and biomarkers of oxidative stress, splenocytes were treated with the test reagents for 48 h at a density of 5 × 10 6 cells/mL in 96-well plates. Culture supernatants were collected and the GSH-Px, SOD, and CAT activities and GSH, MDA, and •OH levels were determined using commercial assay kits. The lower detection limits of the kits were 0.5 U GSH-Px/mL, 0.5 U SOD/mL, 0.04 U CAT/mL, 0.04 U • OH/mL, 0.01 mmol GSH/mL, and 0.01 mmol MDA/mL, respectively.

Western Blotting
Western blotting was performed as previously described in Liu et al. [28]. Cells were harvested and washed with PBS. Proteins were extracted with radio immunoprecipitation assay (RIPA) lysis buffer containing 10 mg/mL phenylmethanesulfonyl fluoride (PMSF) on ice for 30 min. The protein content was quantified and resolved on SDS-PAGE (10-12% gels) and transferred to polyvinylidene fluoride (PVDF) membranes (Bio-Rad, Richmond, CA, USA). The membranes were blocked with 3% bovine serum albumin (BSA) for 1 h at room temperature and incubated with primary antibodies against NOX2, DUOX1, TLR4, and TLR7 at 4 • C, overnight. Following washing with Tris-buffered saline and Tween20 (TBST) three times, the membranes were incubated with HRP-conjugated secondary antibody at room temperature for 1 h. Finally, the membranes were colored with 10 mg 3,3-diaminobenzidine (DAB) solution in 50 mL phosphate buffer (0.03 M) plus 20 µL H 2 O 2 . β-Actin was used as an endogenous control. Bands were analyzed using Bio-Rad Laboratories ver. 4.5 Quantity One software (Hercules, CA, USA).

Statistical Analysis
All data are expressed as the mean ± SD obtained from at least three individual experiments. Statistical analyses were performed using one-way analysis of variance (ANOVA) followed by a post hoc Tukey's test with GraphPad Prism software (Version 6.01, GraphPad, San Diego, CA, USA). p-values < 0.05 and <0.01 were accepted as significant and very significant, respectively.

Effects of WPE on the Cytotoxicity of MT and CPF on Splenic Lymphocyte Subpopulations
Treatment with an appropriate concentration of Con A and LPS induced the proliferation of T and B cells, respectively [31,32]. We used splenocytes stimulated by Con A and LPS as splenic T and B cells. To investigate the effects of WPE on splenic T-and B-cell populations exposed to MT or CPF, we treated splenocytes with MT or CPF with or without WPE for 48 h in the presence of Con A or LPS and evaluated their viability by MTT assay.  (1,5, and 10 µg/mL) of WPE or ellagic acid or proanthocyanidin or quercetin with CPF. Cell viability was evaluated by a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. EA, ellagic acid; PC, proanthocyanidin; QUE, quercetin. Results are presented as mean ± SD of three separate experiments. * p < 0.05 or ** p < 0.01 vs. untreated control; # p < 0.05 or ## p < 0.01 vs. MT or CPF treatment.

Effects of WPE on the Cytotoxicity of MT and CPF on Splenic Lymphocyte Subpopulations
Treatment with an appropriate concentration of Con A and LPS induced the proliferation of T and B cells, respectively [31,32]. We used splenocytes stimulated by Con A and LPS as splenic T and B cells. To investigate the effects of WPE on splenic T-and B-cell populations exposed to MT or CPF, we Nutrients 2020, 12, 616 6 of 20 treated splenocytes with MT or CPF with or without WPE for 48 h in the presence of Con A or LPS and evaluated their viability by MTT assay.
As shown in Figure 2A,B, MT significantly inhibited the proliferation of splenic T cells but not that of splenic B cells, whereas CPF significantly inhibited the proliferation of splenic B cells but not that of splenic T cells. However, WPE (1 µg/mL) significantly attenuated the effects of MT on splenic T cells and the effects of CPF on B cells; the viability of splenic T cells exposed to MT increased from 86.2% to 101.4%, and that of splenic B cells exposed to CPF increased from 82.4% to 102.8% ( Figure 2C,D). To further investigate the effects of WPE on splenic lymphocyte subsets exposed to MT or CPF, we reacted splenocytes with fluorescein isothiocyanate (FITC)-labeled antibodies and assessed the results with flow cytometry. As shown in Figure 3, proportions of CD3 + , CD4 + , and CD8 + T cells were significantly reduced in splenocytes exposed to MT relative to the control; a similar result was obtained for CD19 + B cells exposed to CPF. However, MT did not affect the number of CD19 + B cells, and CPF did not decrease the number of CD3 + , CD4 + , or CD8 + T cells, consistent with the above results. WPE significantly increased the proportion of CD3 + , CD4 + , and CD8 + T cells exposed to MT and the proportion of CD19 + B cells exposed to CPF ( Figure 4A-D). At 1 µg/mL WPE, the CD3 + T-, CD4 + T-, and CD19 + B-cell populations were similar to the controls ( Figure 4A-D). Therefore, WPE is capable of normalizing proportions of splenic T cells exposed to MT or splenic B cells exposed to CPF.
Nutrients 2020, 01, x FOR PEER REVIEW 6 of 20 As shown in Figure 2A,B, MT significantly inhibited the proliferation of splenic T cells but not that of splenic B cells, whereas CPF significantly inhibited the proliferation of splenic B cells but not that of splenic T cells. However, WPE (1 μg/mL) significantly attenuated the effects of MT on splenic T cells and the effects of CPF on B cells; the viability of splenic T cells exposed to MT increased from 86.2% to 101.4%, and that of splenic B cells exposed to CPF increased from 82.4% to 102.8% ( Figure  2C,D). To further investigate the effects of WPE on splenic lymphocyte subsets exposed to MT or CPF, we reacted splenocytes with fluorescein isothiocyanate (FITC)-labeled antibodies and assessed the results with flow cytometry. As shown in Figure 3, proportions of CD3 + , CD4 + , and CD8 + T cells were significantly reduced in splenocytes exposed to MT relative to the control; a similar result was obtained for CD19 + B cells exposed to CPF. However, MT did not affect the number of CD19 + B cells, and CPF did not decrease the number of CD3 + , CD4 + , or CD8 + T cells, consistent with the above results. WPE significantly increased the proportion of CD3 + , CD4 + , and CD8 + T cells exposed to MT and the proportion of CD19 + B cells exposed to CPF ( Figure 4A-D). At 1 μg/mL WPE, the CD3 + T-, CD4 + T-, and CD19 + B-cell populations were similar to the controls ( Figure 4A-D). Therefore, WPE is capable of normalizing proportions of splenic T cells exposed to MT or splenic B cells exposed to CPF. Results are presented as mean ± SD of three separate experiments. * p < 0.05 or ** p < 0.01 vs. untreated control; # p < 0.05 or ## p < 0.01 vs. MT or CPF treatment.

Effects of WPE on Cytokine Production by MT-or CPF-Stimulated Cells
To investigate the effects of WPE on their function, we treated splenic lymphocytes with MT or CPF in the presence or absence of WPE and analyzed cytokine/granzyme production by enzyme-linked Nutrients 2020, 12, 616 7 of 20 immunosorbent assay (ELISA). IL-2 and IFN-γ were used as markers of CD4 + Th1 cells, IL-4 as a marker of CD4 + Th2 cells, granzyme B as a marker of CD8 + T cells, and IL-6 as a marker of B cells. MT significantly suppressed the synthesis of IL-2, IFN-γ, IL-4, and granzyme B but did not affect that of IL-6 ( Figure 5). By contrast, CPF significantly decreased the production of IL-6 but did not affect that of IL-2, IFN-γ, IL-4, or granzyme B ( Figure 5). These results, which are consistent with those for cell viability and flow cytometry, show that MT selectively induces toxicity in T cells and CPF selectively induces toxicity in B cells.  WPE (1 µg/mL) increased the synthesis of IL-2, IFN-γ, IL-4, and granzyme B from 86.0% to 95.5% (392.9 to 436.4 pg/mL), 88.4% to 95.4% (343.1 to 370.4 pg/mL), 87.5% to 97.4% (31.0 to 34.5 pg/mL), and 89.7% to 98.0% (119.0 to 130.1 units), respectively, in T cells exposed to MT relative to the control ( Figure 6A-D). In addition, WPE (1 µg/mL) significantly increased the production of IL-6 by B cells exposed to CPF from 44.6% to 87.1% (27.1 to 53.0 pg/mL) relative to the control ( Figure 6E). Therefore, WPE prevents the MT-or CPF-induced decrease in the production of T-or B-cell-associated cytokines/granzyme by splenic lymphocytes.

Effects of WPE on Cytokine Production by MT-or CPF-Stimulated Cells
To investigate the effects of WPE on their function, we treated splenic lymphocytes with MT or CPF in the presence or absence of WPE and analyzed cytokine/granzyme production by enzymelinked immunosorbent assay (ELISA). IL-2 and IFN-γ were used as markers of CD4 + Th1 cells, IL-4 as a marker of CD4 + Th2 cells, granzyme B as a marker of CD8 + T cells, and IL-6 as a marker of B cells. MT significantly suppressed the synthesis of IL-2, IFN-γ, IL-4, and granzyme B but did not affect that of IL-6 ( Figure 5). By contrast, CPF significantly decreased the production of IL-6 but did not affect that of IL-2, IFN-γ, IL-4, or granzyme B ( Figure 5). These results, which are consistent with those for cell viability and flow cytometry, show that MT selectively induces toxicity in T cells and CPF selectively induces toxicity in B cells.

Effects of WPE on Cytokine Production by MT-or CPF-Stimulated Cells
To investigate the effects of WPE on their function, we treated splenic lymphocytes with MT or CPF in the presence or absence of WPE and analyzed cytokine/granzyme production by enzymelinked immunosorbent assay (ELISA). IL-2 and IFN-γ were used as markers of CD4 + Th1 cells, IL-4 as a marker of CD4 + Th2 cells, granzyme B as a marker of CD8 + T cells, and IL-6 as a marker of B cells. MT significantly suppressed the synthesis of IL-2, IFN-γ, IL-4, and granzyme B but did not affect that of IL-6 ( Figure 5). By contrast, CPF significantly decreased the production of IL-6 but did not affect that of IL-2, IFN-γ, IL-4, or granzyme B ( Figure 5). These results, which are consistent with those for cell viability and flow cytometry, show that MT selectively induces toxicity in T cells and CPF selectively induces toxicity in B cells.

Effects of WPE on Levels of Apoptosis-Associated Proteins
Next we investigated the effects of WPE on levels of apoptosis-associated proteins (Bax, Bcl-2, and p53) with Western blotting. Levels of Bax, Bcl-2, and p53 were significantly increased in MT-exposed T cells and in CPF-exposed B cells. WPE significantly decreased Bax and p53 levels in T cells stimulated by MT and in B cells exposed to CPF ( Figure 7A,C,D,F). However, WPE did not significantly affect the expression of Bcl-2 in cells exposed to MT or CPF ( Figure 7B,E). Therefore, WPE suppresses the MTand CPF-induced apoptosis of splenic lymphocytes. Figure 5. Effect of MT and CPF on cytokine/granzyme production in splenocytes. Splenocytes were cultured for 48 h in the presence of MT (10 −5 M) or CPF (10 −5 M). Levels of interleukin (IL)-2, interferon (IFN)-γ, IL-4, granzyme B, and IL-6 released into culture media were then measured by Enzymelinked immunosorbent assay (ELISA). Results are presented as mean ± SD of three separate experiments. * p < 0.05 or ** p < 0.01 vs. untreated control. Figure 6. Effect of WPE on cytokine/granzyme production in splenocytes exposed to MT or CPF. Levels of (A) IL-2, (B) IFN-γ, (C) IL-4, (D) granzyme B, and (E) IL-6 released into culture media were then measured by ELISA. Results are presented as mean ± SD of three separate experiments. * p < 0.05 or ** p < 0.01 vs. untreated control. # p < 0.05 or ## p < 0.01 vs. MT or CPF treatment.

Effects of WPE on Levels of Apoptosis-Associated Proteins
Next we investigated the effects of WPE on levels of apoptosis-associated proteins (Bax, Bcl-2, and p53) with Western blotting. Levels of Bax, Bcl-2, and p53 were significantly increased in MTexposed T cells and in CPF-exposed B cells. WPE significantly decreased Bax and p53 levels in T cells stimulated by MT and in B cells exposed to CPF ( Figure 7A,C,D,F). However, WPE did not significantly affect the expression of Bcl-2 in cells exposed to MT or CPF ( Figure 7B,E). Therefore, WPE suppresses the MT-and CPF-induced apoptosis of splenic lymphocytes. Figure 6. Effect of WPE on cytokine/granzyme production in splenocytes exposed to MT or CPF. Levels of (A) IL-2, (B) IFN-γ, (C) IL-4, (D) granzyme B, and (E) IL-6 released into culture media were then measured by ELISA. Results are presented as mean ± SD of three separate experiments. * p < 0.05 or ** p < 0.01 vs. untreated control. # p < 0.05 or ## p < 0.01 vs. MT or CPF treatment.

Effects of WPE on the Production of ROS in MT-and CPF-Stimulated Cells
Next we evaluated levels of •OH, MDA, SOD, GSH-Px, CAT, and GSH in MT-and CPF-exposed splenic lymphocytes. As shown in Figure 8, MT significantly increased •OH and MDA levels and markedly decreased SOD, GSH-Px, and CAT activity and GSH content in splenic T cells. CPF markedly increased •OH and MDA levels and significantly decreased SOD, GSH-Px, and CAT activity and GSH content in splenic B cells (Figure 9). Therefore, MT and CPF induce overproduction of ROS and oxidative stress in splenic lymphocytes.

Effects of WPE on the Production of ROS in MT-and CPF-Stimulated Cells
Next we evaluated levels of •OH, MDA, SOD, GSH-Px, CAT, and GSH in MT-and CPF-exposed splenic lymphocytes. As shown in Figure 8, MT significantly increased •OH and MDA levels and Figure 7. Effects of WPE on the expression of apoptosis-associated proteins in MT-exposed T cells and CPF-exposed B cells. The expression of (A) Bax, (B) Bcl-2, and (C) p53 in splenic T cells and (D) Bax, (E) Bcl-2, and (F) p53 in splenic B cells was measured using Western blotting. Results are presented as mean ± SD of three separate experiments. * p < 0.05 or ** p < 0.01 vs. untreated control; # p < 0.05 or ## p < 0.01 vs. MT or CPF treatment.

Effects of WPE on MT-or CPF-Induced NOX and TLR Expression
To determine whether WPE inhibits the overproduction of ROS through NADPH oxidase complexes (NOX), we evaluated the expression of NOX2 and DUOX1 in splenic lymphocytes treated with MT or CPF alone or in combination with WPE. As shown in Figure 10, NOX2 and DUOX1 expression was significantly increased in MT-and CPF-stimulated cells. The NOX2 and DUOX1 overexpression induced by MT or CPF was significantly reduced by WPE; at >1 μg/mL, the effects of Figure 9. Effects of WPE on the oxidative stress parameters in splenic B cells exposed to CPF. Changes in (A) • OH content, (B) MDA content, (C) SOD activity, (D) GSH-Px activity, (E) CAT activity, and (F) GSH content in B cells were measured using specific assay kits. Results are presented as mean ± SD of three separate experiments. * p < 0.05 or ** p < 0.01 vs. untreated control; # p < 0.05 or ## p < 0.01 vs. MT treatment.

Effects of WPE on MT-or CPF-Induced NOX and TLR Expression
To determine whether WPE inhibits the overproduction of ROS through NADPH oxidase complexes (NOX), we evaluated the expression of NOX2 and DUOX1 in splenic lymphocytes treated with MT or CPF alone or in combination with WPE. As shown in Figure 10, NOX2 and DUOX1 expression was significantly increased in MT-and CPF-stimulated cells. The NOX2 and DUOX1 overexpression induced by MT or CPF was significantly reduced by WPE; at >1 µg/mL, the effects of WPE were nonsignificant in a concentration-dependent manner ( Figure 10).  The expression of TLRs is critical for the production of ROS following NADPH oxidase activation [33][34][35]. TLR4 is expressed in T cells [34], and TLR7 is expressed in B cells [36]. As shown in Figure 11, MT markedly increased the expression of TLR4 in T cells, and CPF markedly increased the expression of TLR7 in B cells. Therefore, MT-or CPF-induced overactivation of NOX2 and DUOX1 might be due to overexpression of TLR4 or TLR7 in splenic lymphocytes. However, overexpression of TLR4 or TLR7 was normalized by WPE in cells treated with MT or CPF ( Figure 11). Therefore, WPE inhibits overactivation of NOX2 and DUOX1 by suppressing MT-and CPF-induced overexpression of TLR4 and TLR7 in T and B cells, respectively.

Discussion
We investigated the effects of WPE on MT-and CPF-induced immunotoxicity using mouse splenocytes. The major findings were as follows: First, WPE attenuated the cytotoxicity of MT and CPF by increasing cell viability and recovering splenic lymphocyte subpopulations. Second, WPE significantly restored the production of IL-2, IL-4, IL-6, IFN-γ, and granzyme B in splenic lymphocytes exposed to MT and CPF. Third, WPE prevented MT-and CPF-induced oxidative damage by normalizing levels of GSH, GSH-PX, SOD, CAT, •OH, and MDA. Finally, WPE inhibited NOX2 and DUOX1 overexpression by suppressing the expression of TLR4 and TLR7. Therefore, WPE protects against MT-and CPF-mediated immunotoxicity by modulating TLRx-NOX-ROS.
The immunotoxicity of OPs has been reported [6,7,37,38]. The spleen is an important immune organ and harbors mainly T and B lymphocytes, which are critical for immunity in animals and humans [39]. OPs, such as diazinon, methyl parathion, and fenitrothion, have toxic effects on the spleen and splenic lymphocytes [30,37]. Polyphenols reportedly protect against the toxicity of OPs [40,41]. Curcumin increases the proliferation of blood lymphocytes exposed to parathion [41], and walnut polyphenols regulate proportions of murine splenic T-cell subpopulations in response to fenitrothion [28]. In this study, MT and CPF significantly suppressed the proliferation of T and B cells, which was significantly reversed by WPE.

Discussion
We investigated the effects of WPE on MT-and CPF-induced immunotoxicity using mouse splenocytes. The major findings were as follows: First, WPE attenuated the cytotoxicity of MT and CPF by increasing cell viability and recovering splenic lymphocyte subpopulations. Second, WPE significantly restored the production of IL-2, IL-4, IL-6, IFN-γ, and granzyme B in splenic lymphocytes exposed to MT and CPF. Third, WPE prevented MT-and CPF-induced oxidative damage by normalizing levels of GSH, GSH-PX, SOD, CAT, •OH, and MDA. Finally, WPE inhibited NOX2 and DUOX1 overexpression by suppressing the expression of TLR4 and TLR7. Therefore, WPE protects against MT-and CPF-mediated immunotoxicity by modulating TLRx-NOX-ROS.
The immunotoxicity of OPs has been reported [6,7,37,38]. The spleen is an important immune organ and harbors mainly T and B lymphocytes, which are critical for immunity in animals and humans [39]. OPs, such as diazinon, methyl parathion, and fenitrothion, have toxic effects on the spleen and splenic lymphocytes [30,37]. Polyphenols reportedly protect against the toxicity of OPs [40,41]. Curcumin increases the proliferation of blood lymphocytes exposed to parathion [41], and walnut polyphenols regulate proportions of murine splenic T-cell subpopulations in response to fenitrothion [28]. In this study, MT and CPF significantly suppressed the proliferation of T and B cells, which was significantly reversed by WPE.
Immune-related cytokines play important roles in activating and modulating immune responses and thus are useful for evaluating immune function [21,42]. However, the pesticide pirimiphos-methyl significantly decreases the production of IL-2, IL-4, IL-6, and IFN-γ in mouse spleen [43]; dimethoate significantly decreases synthesis of IL-2, IL-4, and IFN-γ in mouse spleen [44]; and diazinon decreases IL-2, IL-4, IL-10, IL-12, and IFN-γ levels in splenocytes [45]. Polyphenols have profound effects on cytokine secretion [21,23]. In vivo, curcumin increases levels of IFN-γ and IL-1β in Th1 cells, counteracting nicotine-induced toxicity in rats [20]. Similarly, lychee fruit polyphenols decrease the secretion of IL-6 and TNF-α, thus having anti-inflammatory effects in peripheral-blood monocytes [23]. In this study, MT reduced levels of T-cell-related cytokines, and CPF reduced B-cell-related cytokine, in splenocytes. WPE increased the production of T-cell-related cytokines in splenocytes exposed to MT and the production of B-cell-related cytokine in those exposed to CPF, consistent with the MTT assay and flow cytometry results.
Exposure to OPs can result in the overproduction of ROS, leading to oxidative stress [46][47][48]. Diazinon influences the activity of antioxidant enzymes, causing severe damage to macrophages [48]. Pirimiphos-methyl increases the overproduction of ROS in mice [46]. Polyphenols have antioxidant effects, protecting against damage caused by toxic chemicals [17,28,49]. Pomegranate polyphenols decrease the ROS generation and oxidative damage induced by arsenic [50]. WPE is rich in phenolic compounds, such as ellagitannins and flavonoids. The content of ellagic acid and ellagic tannin, the main components of WPE, is 86.54% [28]. Ellagitannins have radical-scavenging activity due to their phenolic hydroxyl groups [51]. Moreover, flavonoids inhibit free-radical chain reactions by acting as chelators of transition metals via their •OH groups [52]. In this study, MT and CPF induced overproduction of ROS and oxidative stress in splenic lymphocytes, as indicated by significant increases in •OH and MDA levels and marked decreases in SOD, GSH-Px, and CAT activity and GSH content. WPE protected against MT-and CPF-induced oxidative damage by maintaining or increasing the activity of several key antioxidants and decreasing levels of •OH and MDA in splenic lymphocytes.
OPs cause apoptosis of diverse cell types by inducing excessive ROS production. Bax (pro-apoptotic), Bcl-2 (anti-apoptotic), and p53 (pro-apoptotic) play important roles in apoptosis [53,54]. Avermectin induces apoptosis by upregulating the Bax/Bcl-2 ratio and mediating the overproduction of ROS in HepG2 cells [55]. Paraoxon and malaoxon induce apoptosis in human pulmonary cells by inducing oxidative stress [56]. Polyphenols prevent apoptosis by altering the expression of apoptosis-related proteins [57]. Strawberry polyphenols suppress the apoptosis of HepG2 cells by decreasing the intracellular ROS level [58]. Kaempferol upregulates the expression of Bcl-xL and downregulates that of p53 and Bax by inhibiting extracellular regulated protein kinases (ERK), NF-κB, and MyD88 expression in human ovarian cancer cells [59]. In this study, MT and CPF induced apoptosis of splenic lymphocytes by significantly increasing Bax and p53 levels, and WPE prevented apoptosis by restoring the expression of Bax and p53.
TLRs play an important role in immunity via their link with NADPH oxidase homologues [33]. Stimulant recognition by TLRs stimulates NADPH oxidase-mediated ROS formation, an important component of cellular regulation [28]. The expression of TLRs is critical for the production of ROS following NADPH oxidase activation [33][34][35]. TLR4 is a pathogen-associated molecular pattern receptor expressed in phagocytes and T cells [66]. NOX2 is activated by the overexpression of TLR4, a key regulator of innate immunity [34]. LPS-induced activation of TLR4 in vitro enhances generation of ROS by NOX2 in T cells [35]. In addition, TLR4 ligands increase NOX2 expression in dendritic cells [45]. TLR7 is present and functional in B lymphocytes, in which it regulates NADPH oxidase expression and ROS production [47]. TLR7 ligands trigger NADPH oxidase activation, resulting in a high level of ROS in B cells [47]. Influenza A virus infection increases NOX2 oxidase-dependent oxidative stress by upregulating the expression of TLR7 [67]. However, polyphenols decrease the overexpression of TLRs and normalize the level of NADPH oxidase [28,35]. Polyphenols from Antirhea borbonica decrease NOX2-mediated ROS generation in LPS-exposed adipocytes by reducing the expression of TLRs [68]. Similarly, curcumin suppresses LPS-induced, NOX-mediated ROS production by inhibiting TLR4 signaling in rat vascular smooth muscle cells [35]. In this study, MT and CPF caused overexpression of TLR4 and TLR7 in T and B cells, respectively. WPE decreased overexpression of TLR4 in T cells and overexpression of TLR7 in B cells, which suggests that its normalization of NOX2 and DUOX1 was mediated by the regulation of TLR4 in splenic T cells and TLR7 in splenic B cells.

Conclusions
MT and CPF affect the proliferation of, and cytokine/granzyme production by, splenic T and B lymphocytes, respectively, in vitro. WPE protects against MT-and CPF-induced cytotoxicity by inhibiting their effects on cell viability and cytokine/granzyme production in vitro. The protective effects of WPE in MT-or CPF-exposed splenic T and B cells are mediated by the inhibition of oxidative stress via suppression of the activation of NOX2 and DUOX1, the latter caused by downregulation of TLR4 and TLR7, respectively. Further studies are needed to identify the mechanisms underlying the effects of MT and CPF on splenic T and B lymphocytes and the protective effects of WPE.

Conflicts of Interest:
The authors declare no conflict of interest.