Resveratrol pretreatment mitigates LPS-induced acute lung injury by regulating conventional dendritic cells’ maturation and function

2.1 Animals and agents

Six- to eight- weeks-old female C57BL/6 (H-2^b) mice were purchased from experimental animal center of Chinese Academy of Science. All mice were bred and maintained under pathogen-free conditions. All mice were group housed under a 12 h light/dark cycle with ad libitum access to food and water. All mice were euthanized via carbon dioxide inhalation. RES was purchased from Sigma (R5010, USA) and dissolved in DMSO.

1. Ethical approval: The research related to animal use has been complied with all the relevant national regulations and institutional policies for the care and use of animals and was approved by the Care and Use of Laboratory Animals (Ministry of Health, China, 1998) and the guidelines of the Laboratory Animal Ethical Committee of Xuzhou Medical University (Assurances No. L20190226366).

2.2 Mouse model of LPS-induced ALI

The general procedure modeling LPS-induced ALI has been described in previous study [12]. Briefly, mice were anesthetized and placed in a supine position upon a board with head tilted at 37°C. LPS (055:B5, Sigma, USA) was dissolved in 0.9% of saline and intratracheally injected to the trachea at a dosage of 5 mg/kg [28] with a 22-gauge feeding needle. Then, the mice were placed in a vertical position and rotated for 30 s to distribute the LPS evenly within the lungs. Mice in the control group received the same volume of 0.9% of saline and manipulations. All mice were euthanized at 24 h after LPS or saline administration. For mice survival assay, mice survival was monitored daily up to ten days post-challenge.

2.3 Drug treatment

For in vivo assay, RES was administered orally using a 30 mm oral gavage needle at 100 mg/kg in a total volume of 100 μL in an appropriate vehicle of 1% of carboxymethyl cellulose (CMC), a dose established in previous studies [29,30]. RES was given daily for seven days prior to LPS administration for ALI model. For in vitro assay, BMDCs were generated as described below in cell culture section. 0–100 μM of RES was added to the culture medium on initial day, and 500 ng/mL of LPS (055:B5, Sigma, USA) was added to the culture medium on day six. 48 h later, BMDCs were harvested for future experiments [31]. In some experiments, methylthiazolyldiphenyl-tetrazolium bromide (MTT) (C0009S, Beyotime, China) assay was performed to evaluate the viability and cytotoxicity of RES pretreated BMDCs. The schematic diagram of the experimental protocol is shown in Figure 1.

Figure 1

Schematic diagram of experimental protocol.

2.4 Histologic examination

Lung tissues from the mice were removed after 24 h of LPS administration and were fixed in 10% of phosphate-buffered formalin, paraffin-embedded, sectioned, and stained with hematoxylin and eosin (HE), and examined by optical microscope (DM3000b, Leica, Germany) with 200× magnifications. The histopathological changes in mice lung injury were compared quantitatively by calculating the histopathological scores [32], which were scored based on the assignment of one point for each of the following parameters: 1 = Alveolar hyperemia, 2 = Hemorrhage, 3 = Interstitial or leukocyte infiltration or aggregation, and 4 = Alveolar septal thickening. Two independent researchers scored separately in a blinded manner.

2.5 Lung wet/dry (W/D) weight ratios

The middle lobe of right lung was excised and the wet weight was recorded. The lung was then placed in an incubator at 80°C for at least 72 h to obtain the dry weight. The ratio of wet lung to dry lung was calculated.

2.6 MPO activity

Briefly, lung tissue samples were thawed and homogenized in myeloperoxidase (MPO) assay buffer (Abcam). The samples were then freeze-thawed and centrifuged at 12,000g for 10 min at 4°C. The enzyme activity was tested by MPO activity Kit (ab105136, Abcam, UK) according to manufacturer’s instructions using a microplate reader (Biotek, USA). Results were expressed as MPO units per gram of tissue.

2.7 Cytokines measurement

The BALF was collected at indicated time by using three consecutive instillations of 1 mL of PBS at room temperature. The collected BALF of 1,500g was centrifuged at 4°C for 5 min, and the supernatants were stored at −80°C for estimation of cytokine levels. proinflammatory (Th1)- and anti-inflammatory (Th2)-related cytokines [33] were measured by ELISA (BioLegend, USA) kits according to the manufacturers’ instructions.

2.8 Isolation of pulmonary hematopoietic cells

The pulmonary hematopoietic cells’ isolation procedure has been described in our previous study [34]. Briefly, lungs were harvested after perfusion with 10 mL of cold PBS. Tissues were minced and digested in Hank’s buffer containing Liberase TM (05401127001, Roche, Switzerland) and DNase I (B00003, Roche, Switzerland). Single cell suspension was collected by filtering through a 70 μm cell strainer. In some experiments, lymphocytes were further purified using 40% of Percoll gradient centrifugation.

2.9 Flow cytometry and cell sorting

Antibodies (Abs) were purchased from BioLegend (USA) or eBioscience (USA). Abs used included anti-CD45(A20), anti-CD4(RM4-5), anti-CD11b(M1/70), anti-CD11c(N418), anti-F4/80(BM8), anti-CD25(3C7), anti-CD80(16-10A1), anti-CD86(GL-1), anti-MHCII (M5/114.15.2), anti-ILT3(H1.1), anti-IL10(JES5-16E3), anti-IL12(C15.6), anti-IL17(9B10), anti-Foxp3(FJK-16s), and isotype control antibody. For DCs staining, prepared lung mononuclear cells (MNCs) and BMDCs were staining F4/80 first, then F4/80⁻ cells were further stained with CD11b and CD11c, since DCs express the marker CD11c, but not the marker F4/80 on the cell surface [14]. Intracellular staining of transcription factors was performed using Foxp3 Fix/Perm Kit (Thermo, USA) according to the manufacturer’s instructions. Flow cytometric analysis was performed on FACSCanto II (BD Bioscience, USA). Cell sorting was performed using an FACSAria III (BD Biosciences, USA).

2.10 Cell culture

BMDCs were generated as described previously [35]. Briefly, murine bone marrow from the femura and tibiae of C57BL/6 mice were flushed and depleted of red blood cells (RBC) by ACK lysis buffer (Sigma, USA). Cells (1 × 10⁶ cells/mL) were grown in 100 mm dishes in RPMI 1640 containing 10% of FBS, l-glutamine, nonessential amino acids, sodium pyruvate, penicillin–streptomycin, HEPES, and 2-ME (Sigma, USA). rmGM-CSF (40 ng/mL, AF31503, PeproTech, USA), rmIL-4 (40 ng/mL, 21414, PeproTech, USA), and 0–100 μM RES were added to the culture medium in a humidified 5% CO₂ incubator at 37°C on day 0; on day six, non-adherent cells were washed out, adherent cell clusters were harvested, and purified with anti-CD11c beads (Miltenyi Biotec, Germany). Subsequently, 500 ng/mL of LPS was added to the purified cells for 48 h. On day eight, cells were collected for analysis or future experiments. For RES-treated BMDCs co-cultured with sorted CD4 from ALI mice, two kinds of cells (ratio 1:1 or 3:1) were co-cultured in the medium containing rmIL-2 (5 ng/mL, 21212, PeproTech, USA), rhTGF-β (5 ng/mL, 10021 C, PeproTech, USA), and rmIL-6 (20 ng/mL, 21616, PeproTech, USA) for three days and then the cells were stained with antibodies against CD25, Foxp3, IL-17, or IL-10 using intracellular or surface staining following flow cytometry analysis. In some experiments, sILT3 (10 μg/mL, 328978, ACROBiosystems, USA) were added into the co-cultured medium following flow cytometry analysis.

2.11 Adoptive cell transfer

C57BL/6 (H-2^b) mice were immediately injected with 1 × 10⁶ RES-pretreated BMDCs via tail veil 1 day before LPS challenge and then the mice were euthanated after 24 h for future experiments.

2.12 Statistical analysis

Statistical analysis were performed using GraphPad Prism 6 Software (La Jolla, CA). Data were presented as mean values ± SEM and statistically analyzed with two-tailed independent Student’s t-test (two groups). For multiple groups, 1-way ANOVA was used for each experiment. For in vivo experiments, groups of 5–10 mice were used per experimental group. For in vitro assays, all experiments were performed in triplicate and one representative experiment was presented. For mice survival, Kaplan–Meyer analysis with log-rank test was used. The level of statistical significance was set at *p < 0.05, **p < 0.01, and ***p < 0.001.

3.1 RES alleviated LPS-induced ALI

In order to investigate the protective role of RES in LPS-induced ALI, the mice were challenged with LPS for 24 h, then lung histological changes were evaluated in mice with or without RES oral pretreatment for seven days. As shown in Figure 2a, the mice without RES pretreatment showed significant histological deterioration according to the lung HE stained section, such as alveolar wall thickness, edema, and inflammatory cell infiltration, which were greatly reversed by 100 mg/kg of RES-pretreated. No obvious cytotoxicity has been found in mice receiving 100 mg/kg of RES alone group (Figure 2a and b). Meanwhile, 25 mg/kg of RES had no protective effects against ALI, since lung of the mice that received 25 mg/kg of RES treatment had no significant difference compared with no treatment ALI mice (Figure 2a and b). However, lung of mice that received 50 mg/kg of RES treatment had a slight but not a significant recovery than that of no treatment or 25 mg/kg of RES treatment mice, lung of mice that received 100 mg/kg of RES treatment had a significant improvement than that of 50 mg/kg treatment mice group (Figure 2a and b). In accordance, the lung injury score system demonstrated that 100 mg/kg of RES pretreatment could efficiently attenuate the severity of ALI upon exposure to LPS for 24 h compared with ALI mice without RES pretreatment (Figure 2b). Thus, mice which received 100 mg/kg of RES treatment were used for further study. Furthermore, as a lung vascular permeability indicator, the lung W/D weight ratio significantly decreased in RES-pretreated mice compared with untreated mice, indicating that RES led to less lung edema (Figure 2c). Next, BALF cells and protein concentration were collected and determined as an index of integrity of epithelial basement membrane. Result showed that BALF cells and protein level statistically decreased in RES-pretreated mice compared with ALI mice alone, suggesting that RES had an inhibitory capacity of preventing cells and protein leakage (Figure 2d and e). Finally, in LPS-challenged mice, MPO activity in lung tissue was also prominently reduced with RES pretreatment, whereas mice without RES pretreatment had markedly raised MPO activity (Figure 2f). Taken together, RES could attenuate LPS-induced ALI.

Figure 2

The alleviation of LPS-induced ALI by oral pretreatment with RES. RES was administrated orally daily for seven days prior to LPS administration for ALI model. Lung was harvested 24 h after LPS intratracheal administration. (a) The representative pulmonary HE section is shown for each group (magnification:200×); (b) pulmonary histopathological scores are determined in six groups at 24 h; (c) the wet lung-to-dry weight ratio (W/D ratio) in three groups at 24 h; (d and e) total cells and proteins in BALF were analyzed in three groups at 24 h; (f) lung MPO activity is calculated in three groups at 24 h. Data are from one representative experiment of three performed and presented as the mean value ± SEM, 1-way ANOVA was used for pulmonary histopathological scores and W/D ratio, n = 10; for cells and proteins in BALF and MPO activity, n = 5, *P < 0.05.

3.2 RES modulated inflammatory responses in LPS-induced ALI

To address the effect of RES on pulmonary inflammation in LPS-induced ALI, the pro/anti-inflammatory cytokines in BALF were determined 24 h post LPS challenge. The level of proinflammatory cytokines, including TNF-α, IL-2, IL-6, and IL-12p70, were strikingly decreased (Figure 3a), whereas the level of anti-inflammatory cytokines, including TGF-β, IL-10, IL-13, and IL-33 were notably increased (Figure 3b) in RES-pretreated mice compared with ALI group. These data suggested that RES inhibited Th1, but promoted Th2 immune responses in LPS-induced ALI.

Figure 3

The profiles of decreased proinflammatory (Th1)-related cytokines but increased anti-inflammatory (Th2)-related cytokines 24 h after LPS challenged by RES pretreatment. The mice BALF sample were collected within 3 treatment groups at indicated time. Th1- and Th2- related cytokines were measured by ELISA kits. (a) Decreased Th1-related cytokines including TNF-α, IL-2, IL-6, and IL-12p70 were observed. (b) Increased Th2-related cytokines containing TGF-β, IL-10, IL-13, and IL-33 were determined. Data were from one representative experiment of three performed and presented as the mean value ± SEM (n = 5), t-test was used, *P < 0.05.

3.3 RES regulated pulmonary cDC maturation and function in LPS-induced ALI mice

Study has shown that pulmonary cDC was significantly increased in the pathological process of ALI [14]. Hence, we studied the effects of RES on cDCs in our model. First, we tested whether RES pretreatment could arrest pulmonary cDC expansion in LPS-induced ALI. Single cell suspension from lung and spleen were prepared for analysis at 24 h post LPS challenge, since splenic and pulmonary cDCs rapidly increased and maintained for 24 h [19]. Although significant pulmonary cDC expansion was observed in LPS-challenged mice compared with control mice, RES pretreatment led to slightly decreased pulmonary cDCs compared with that of non-pretreated ALI mice (Figure 4a), indicating that RES pretreatment had no significant impact on pulmonary cDC expansion. In line with pulmonary cDCs, no change in the percentage of splenic cDCs occurred (Figure 4a) 24 h after LPS exposure.

Figure 4

Pretreated RES has no influence on cDCs expansion, but regulated pulmonary and splenic cDCs maturation and function. 24 h after LPS exposure, cDCs’ responses were evaluated in differential treatment mice groups. (a) The representative flow cytometry profile and frequency of cDCs (CD11b⁺CD11c⁺) response in lung and spleen upon LPS challenge. Plots were pregated on CD45 cells. (b–d) The representative flow cytometry profile and frequency of co-stimulatory molecules (CD80 and CD86) and MHC II in lung and spleen upon LPS challenge. (e) The representative flow cytometry profile and frequency of inhibitory molecule ILT3 in lung and spleen upon LPS challenge. (f) The representative flow cytometry profile and frequency of IL-12 and IL-10 secretion in lung upon LPS challenge. Plots were pregated on cDCs cells (from b to f). Data were from one representative experiment of three performed and presented as the mean value ± SEM (n = 5), 1-way ANOVA was used, *P < 0.05, **P < 0.01, and ***P < 0.001, n.s means no significance.

Several reports have demonstrated that the maturation of pulmonary cDCs controlled the pathogenesis of LPS-induced ALI [13,19]. Moreover, RES could influence monocytes to DCs differentiation, maturation, and function in vitro [35]. Meanwhile, pulmonary cDC expansion was unaffected by RES pretreatment, hence we next explored whether the maturation and function status of cDCs could be modulated by RES pretreatment in LPS-exposed mice. The co-stimulatory molecules CD80, CD86 and MHC II expression level were associated with maturation status [36]. Accordingly, we checked these molecules’ expression on pulmonary and splenic cDCs. Interestingly, the co-stimulatory molecules CD80 and CD86, and MHC II had higher expressions on both the pulmonary and splenic cDCs in LPS-challenged mice than those in control mice (Figure 4b–d). Importantly, RES pretreatment dramatically suppressed these molecules expression on both the pulmonary and splenic cDCs upon LPS challenge (Figure 4b–d). In addition, RES pretreatment led to increased ILT3 expression on both the pulmonary and splenic cDCs (Figure 4e), which was known to be a DC tolerogenic marker with a potency to induce regulatory T cells (Treg) and to regulate the balance Th cells’ immune responses [37]. Consistent with the BALF inflammatory level in Figure 3, RES-pretreated pulmonary cDCs secreted less IL-12, but more IL-10 than those of ALI mice (Figure 4f). Summarily, these data indicated that RES pretreatment might have no influence on cDC expansion, while it could regulate pulmonary cDCs maturation and function in LPS-induced ALI mice.

3.4 RES regulated the maturation and function of BMDCs in vitro

Due to the maturation and function of pulmonary cDC modulated by pretreated RES in vivo, we subsequently explored whether RES treatment could control the maturation and function of BMDCs in vitro. BMDCs were cultured and their responses to RES treatment in vitro were tested. First, we measured the effects of RES on BMDC viability and cytotoxicity by MTT. Various concentrations of RES were added into cultured BMDCs medium on the initial day and for eight days. Data showed that the frequency of cultured BMDCs slightly decreased compared with the untreated BMDCs in vitro by the administration of relatively low doses of RES ( ≤ 50 μM) (Figure 5a). However, at high dose of RES (100 μM), obvious cytotoxicity was detected so that further experiments were performed with RES at the concentration of ≤50 μM. Accordingly, RES (50 μM) did not simply obstruct the proliferation of DCs in vitro, in line with our in vivo and previous data [38]. Thus, we checked the maturation markers’ expression, which significantly decreased in vivo, on BMDCs on day eight by flow cytometry. As expected, CD80, CD86, and MHC II dramatically decreased in RES-treated BMDCs compared with untreated cells (Figure 5b). Subsequently, the modulation of Th1 and Th2-skewed cytokines by RES on BMDCs was confirmed by IL-12 and IL-10 expressions and secretion, since lower IL-12⁺ cells frequency (Figure 5c) and lower supernatant of IL-12 (Figure 5d), but higher IL-10⁺ cells frequency (Figure 5e) and higher supernatant of IL-10 (Figure 5f) were found by cultured BMDCs in the presence of RES at various concentrations for eight days than those of control counterpart. Interestingly, the tolerogenic marker ILT3, which increased in the ALI mice model, also increased in a dose-dependent manner in vitro (Figure 5g). Taken together, these data revealed that the maturation and function of BMDCs could also be regulated by RES treatment in vitro.

Figure 5

Regulation of cultured BMDCs maturation and function by RES. RES was added into BM cells on initial day to regulate BMDCs maturation combined with 40 ng/mL rmGM-CSF and 40 ng/mL rmIL-4 for 6 days, then 500 ng/mL LPS was administrated for 48 h to induce maturation. (a) The proliferation and cytotoxicity of RES on BMDCs were determined by MTT. Differential concentrations of RES wereused (0, 12.5, 25, 50, and 100 μM). (b) The representative flow cytometry profile and frequency of costimulatory molecules (CD80 and CD86) and MHC II on cultured BMDCs with various concentrations of RES treatment. (c and e) The representative flow cytometry profiles of IL-12⁺ and IL-10⁺ cells on cultured BMDCs upon LPS stimulation with RES treatment (50 μM); (d and f) the supernatant of cultured BMDCs upon LPS stimulation with various concentrations of RES treatment was determined by ELISA. (g) The representative flow cytometry profile and frequency of ILT3 on cultured BMDCs with various concentrations of RES treatment. Data were from one representative experiment of three performed and presented as the mean value ± SEM (n = 5), 1-way ANOVA was used, *P < 0.05 and **P < 0.01, n.s means no significance.

3.5 BMDCs by RES pretreatment regulated CD4 T cells producing more IL-10, but less IL-17 in vitro

The balance between Th1 and Th2, which are crucial to maintain the homeostasis of immune system, is one of causes for the development and progression of ALI [39]. The balance between Treg and Th17 cells has been confirmed to be involved in ALI/ARDS [40]. Our result also indicated that ILT3, which has an ability to modulate Th cell immune responses, was significantly upregulated in cDCs. Thus, to answer whether RES treatment could affect BMDCs mediated CD4 T cells toward Treg or Th17 polarization in vitro, we co-cultured RES-treated BMDCs with sorted splenic CD4 T cells from ALI mice for three days. Results showed that BMDCs did not affect CD4 T cells toward Treg polarization, since the increase in CD25⁺Foxp3⁺ cells on CD4 T cells was not significant (Figure 6a), but it promoted CD4 T cells secreting more IL-10 but less IL-17 compared with its untreated counterpart (Figure 6b). To our surprise, even more IL-10 but less IL-17 secretion on sorted CD4 T cells from ALI mice in the co-culture system was regulated by the administration of sILT3 protein (10 μg/mL) (Figure 6c), indicating that highly expressed ILT3 on BMDCs might mediate CD4 T cells producing IL-10 and IL-17. Hence, these results showed that BMDCs by RES treatment might have no impact on CD4 T cells toward Treg polarization, but could mediate more IL-10 and less IL-17 production on CD4 T cells.

Figure 6

Regulation of CD4 T cells-driven IL-10 and IL-17 secretion by RES treated BMDCs. RES (50 μM) treated BMDCs were co-cultured with sorted CD4 T cells from ALI mice with rmIL-2 (5 ng/mL), rhTGF-β (5 ng/mL), and rmIL-6 (20 ng/mL) for three days. (a) The representative flow cytometry profile and frequency of CD25⁺Foxp3⁺ cells in co-cultured system (DCs:CD4 T = 1:1 or 3:1, respectively). Plots were pregated on CD4⁺ cells. (b) The representative flow cytometry profile and frequency of CD4⁺IL-10⁺ cells and CD4⁺IL-17⁺ cells from co-cultured system (DCs:CD4 T = 3:1). (c) The representative flow cytometry profile and frequency of CD4⁺IL-10⁺ cells and CD4⁺IL-17⁺ cells from co-cultured system (DCs:CD4 T = 3:1) in the presence of sILT3 protein (10 μg/mL). Data were from one representative experiment of three performed and presented as the mean value ± SEM (n = 5), t-test was used, *P < 0.05 and **P < 0.01, n.s means no significance.

3.6 RES-treated BMDCs enhanced the recovery of ALI

To determine whether RES-treated BMDCs had a potential protective role in ALI mice, C57BL/6 mice were adoptively transferred with 1 × 10⁶ RES-treated BMDCs through the tail vein one day before LPS challenge. We first observed the survival rate of ALI mice that received RES-treated BMDCs. Data showed that the survival rate (80%) of mice in tolerogenic BMDCs received group was higher than that (54%) of the unreceived group (Figure 7a). We then used a histological evaluation of lungs to confirm the effect of RES-treated BMDCs in ALI mice. Lung section showed a severity of alveolar wall thickness and inflammatory cell infiltration without treatment at 24 h post LPS challenge, whereas the histological damage of lungs of mice which received RES-treated BMDCs were moderated (Figure 7b). W/D ratio and BALF cell and protein levels in BMDC-transferred mice were improved compared with those of untreated mice (Figure 7c and d).

Figure 7

Adoptive transfer RES treated BMDCs attenuates ALI. 1 × 10⁶ RES treated BMDCs i.v one day before LPS-challenged ALI mice. (a) The survival rate of mice was observed until day 10 following LPS challenge. (b) Pulmonary histopathological scores were determined at 24 h after LPS challenge (magnification: 200×). (c) The wet lung-to-dry weight ratio (W/D ratio) was monitored at 24 h after LPS challenge. (e) Total cells and proteins in BALF were analyzed at 24 h after LPS challenge. (e) The representative flow cytometry profile and frequency of CD11b⁺CD11c⁺ cells in lung and spleen from ALI mice. (f) The representative flow cytometry profile and frequency of pulmonary CD11c⁺IL-10⁺ cells from ALI mice. (g) The representative flow cytometry profile and frequency of peripheral CD4⁺IL-10⁺ and CD4⁺IL17⁺ cells from ALI mice. Data were from one representative experiment of three performed and presented as the mean value ± SEM (n = 5), 1-way ANOVA was used, for survival assay, n = 15, Kaplan–Meyer analysis with log-rank test was used, *P < 0.05, **P < 0.01, and ***P < 0.001.

Mechanistically, compared with untreated mice, the BMDC-transferred mice showed a higher proportion of cDCs in lung and spleen (Figure 7e), which produced more IL-10 (Figure 7f). Additionally, the mice that received BMDCs had a great increase in the proportion of IL-10 producing CD4 T cells, but a sharp decrease in the proportion of IL-17 producing CD4 T cells in the lung compared with untreated mice (Figure 7g). These data indicated that BMDCs by RES treatment could improve the recovery of ALI.