Nrf2 activation mediates tumor-specific hepatic stellate cells-induced DIgR2 expression in dendritic cells

Our previous studies discovered that tumor-specific hepatic stellate cells (tHSCs) induced dendritic cell-derived immunoglobulin receptor 2 (DIgR2) expression in bone marrow-derived dendritic cells (mDCs), inhibiting splenic T cell activation. The current study aims to explore the underlying mechanism of DIgR2 expression by focusing on Nrf2 (nuclear-factor-E2-related factor 2) signaling. We show that tHSCs co-culture induced significant Nrf2 signaling activation in mDCs. The latter was evidenced by Nrf2-Keap1 disassociation, Nrf2 protein stabilization, accumulation and nuclear translocation. Expression of Nrf2-dependent genes, including heme oxygenase-1 (HO-1) and NAD(P)H:quinone oxidoreductase 1 (NQO1), were detected in tHSCs-co-cultured mDCs. Importantly tHSCs-induced DIgR2 expression was blocked by Nrf2 shRNA or knockout (KO, by CRISPR/Cas9 method). Conversely, forced activation of Nrf2, by Keap1 shRNA or the Nrf2 activators (3H-1,2-dithiole-3-thione and MIND4-17), induced significant DIgR2 expression. tHSCs stimulation induced reactive oxygen species (ROS) production in mDCs. Conversely, ROS scavengers inhibited tHSCs-induced ROS production, Nrf2 activation and DIgR2 expression in mDCs. Significantly, tHSCs inhibited production of multiple cytokines (CD80, CD86 and IL-12) in mDCs, reversed by Nrf2 depletion. Moreover, Nrf2 shRNA or KO attenuated splenic T cell inhibition by tHSCs-stimulated mDCs. Together, we conclude that Nrf2 activation mediates tHSCs-induced DIgR2 expression in mDCs.


RESULTS tHSCs co-culture induces Nrf2 signaling activation in mDCs
Our previous studies have shown that tHSCs co-culture induces DIgR2 expression in bone marrow-derived DCs (mDCs) [16,17]. The current study aims to test the potential involvement of Nrf2 signaling in the process. First, we tested whether tHSCs co-culture activated Nrf2 signaling activation in mDCs. As described [16,17], mDCs were co-cultured with primary tHSCs (mDCs to tHSCs ratio, 20: 1). A co-immunoprecipitation ("Co-IP") assay was performed to test the association between Nrf2 and its suppressor protein Keap1 [22,23]. As shown, Nrf2 immunoprecipitated with Keap1 in the untreated control mDCs ( Figure 1A). Following co-culture with tHSCs, Nrf2 was disassociated with Keap1 ( Figure 1A). Furthermore, Nrf2 protein was accumulated in the cytoplasm of mDCs ( Figure 1B). Additionally, analyzing nuclear lysate proteins in mDCs confirmed that stabilized Nrf2 translocated to cell nuclei following tHSCs stimulation ( Figure 1C). These results show that tHSCs co-culture induced Nrf2-Keap1 disassociation, Nrf2 protein accumulation and nuclear translocation in mDCs ( Figure 1A-1C).
Following nuclear translocation, Nrf2 will bind to ARE to promote transcription and expression of multiple anti-oxidant and detoxifying enzymes, including heme oxygenase-1 (HO-1) and NAD(P)H:quinone oxidoreductase 1 (NQO1) [22,23]. We show that HO1 and NQO1 mRNA ( Figure 1D) and protein ( Figure 1E) levels were significantly increased in tHSCs-stimulated mDCs. In line with our previous studies [16,17], DlgR2 mRNA and protein levels were increased as well in mDCs with tHSCs stimulation ( Figure 1D and 1E). The Nrf2 mRNA levels were however unchanged ( Figure  1D), indicating that Nrf2 protein upregulation was due to post-transcriptional regulation (Nrf2-Keap1 disassociation). These results suggest that tHSCs coculture induced Nrf2 signaling activation in mDCs.

Nrf2 activation is required for tHSCs-induced DIgR2 expression in mDCs
Next, we tested the functional activity of Nrf2 activation in tHSCs-induced DIgR2 expression in mDCs. To block Nrf2 activation, genetic strategies [24,25] were utilized. First, the Nrf2 shRNA lentiviral particles were added to mDCs, with puromycin selection the stable cells were established. Additionally, the CRISPR/Cas9 gene editing method was employed to complete knockout (KO) Nrf2 in mDCs (see Methods). Testing Nrf2 mRNA and protein expression confirmed that Nrf2 was depleted in stable mDCs with Nrf2 shRNA or Nrf2 KO construct, even after tHSCs stimulation (Figure 2A and 2B). tHSCs-induced HO1 and NQO1 expression, the Nrf2 target genes [26,27], was blocked by Nrf2 shRNA or KO (Figure 2A and 2B). Importantly, Nrf2 shRNA or KO reversed tHSCs-induced DIgR2 mRNA and protein expression in mDCs (Figure 2A and 2B). These results indicate that Nrf2 activation is required for tHSCs-induced DIgR2 expression in mDCs.

tHSCs-induced Nrf2 activation in mDCs contributes to inhibition on cytokines
Our previous studies have shown that tHSCs co-culture significantly inhibited production of multiple cytokines in mDCs. Such actions were abolished with DIgR2 depletion. Since Nrf2 activation is required for DIgR2 expression, we proposed that Nrf2 depletion should abolish tHSCs-induced inhibition on cytokines in mDCs. In line with our previous findings, in mDCs expression of CD80 (one key surface co-stimulatory molecule, Figure  4A), CD86 (another surface co-stimulatory molecule, Figure 4B) and IL-12 (DC activation marker cytokine, Figure 4C) was decreased following tHSCs co-culture. Significantly, Nrf2 shRNA or KO alleviated tHSCsmediated inhibition of the cytokines in mDCs ( Figure  4A-4C). ELISA results further show that productions of CD80 ( Figure 4D), CD86 ( Figure 4E) and IL-12 The bone marrow-derived dendritic cells (mDCs) were cocultured with/without tumor HSCs (tHSCs; mDCs to tHSCs ratio, 20: 1) for applied time, Nrf2-Keap1 association was tested by the coimmunoprecipitation ("Co-IP") (A); Expression of listed proteins in cytoplasm lysates (B and E) and nuclei lysates (C) were tested by Western blotting; Expression of listed mRNAs was tested by qPCR (D). Listed proteins were quantified and normalized to the loading control (A-C, E). Lamin-B1 is a nuclear marker protein (C). "Tubulin" stands for the loading control β-Tubulin (Same for all Figures). Data are presented as the mean ± standard deviation (n=5). "Ctrl" stands for mDCs only (no tHSCs stimulation). * P < 0.05 vs. "Ctrl" group. The experiments in this figure were repeated four times, and similar results were obtained. AGING (mDCs), with Nrf2 shRNA ("sh-Nrf2") or Nrf2 KO construct ("ko-Nrf2"), as well as the parental control mDCs ("Pare"), were co-cultured with tumor HSCs (tHSCs; mDCs to tHSCs ratio, 20: 1) for applied time, expression of listed genes was shown (A and B). Expression of listed genes in stable mDCs with Keap1 shRNA ("sh-Keap1") or the parental control mDCs ("Pare") was shown (C and D). Control mDCs were treated with 3H-1,2-dithiole-3-thione (D3T, 25 μM), MIND4-17 (10 μM) or vehicle control (0.25% DMSO) for applied time, listed genes were shown (E and F). Data are presented as the mean ± standard deviation (n=5). "Ctrl" stands for mDCs only. * P < 0.05 vs. "Pare" cells (A and C). * P < 0.05 vs. "DMSO"-treated cells (E). The experiments in this figure were repeated three times, and similar results were obtained. AGING ( Figure 4F) were reduced in tHSCs-stimulated mDCs. Such actions were inhibited with Nrf2 silencing or KO ( Figure 4D-4F). These results imply that tHSCsinduced Nrf2 activation in mDCs contributes to inhibition on cytokines.

Nrf2 activation mediates splenic T cell inhibition by tHSCs-stimulated mDCs
Studies from our group [16,17] and others have demonstrated that DC-derived DIgR2 binds to its receptor in T cells, suppressing T cell functions. Using the described protocol [16,17], the splenic T cells were co-cultured with mDCs (T cells to mDCs ratio, 20:1). Ovalbumin II (OVA-II) peptide cytotoxic T lymphocyte (CTL) assay results demonstrated that tHSCs-stimulated mDCs can significantly inhibit the CTL activity of splenic T cells. Furthermore, lipopolysaccharides (LPS)induced interferon-γ (IFN-γ) production in splenic T cells was largely inhibited after co-culture of tHSCsstimulated mDCs ( Figure 5B). Importantly, Nrf2 shRNA or KO in mDCs (see Figure 2) almost reversed splenic T cell inhibitions by tHSCs-stimulated mDCs ( Figure 5A and 5B). These results suggest that Nrf2 activation is important for splenic T cell inhibitions by tHSCs-stimulated mDCs.

DISCUSSION
Immune evasion of HCC cells [7][8][9] is vital for cancer progression [9,35]. tHSCs could be an important cause of T-cell hypo-responsiveness in HCC. Studies have shown that B7-H1 (a key member of the B7 family of co-stimulatory molecules) upregulation in tHSCs can Figure 3. tHSCs-induced Nrf2 activation and DIgR2 expression in mDCs is associated with reactive oxygen species (ROS) production. The bone marrow-derived dendritic cells (mDCs) were pretreated for 1h with N-acetylcysteine (NAC, 500 μM) or MnTBAP (40 μM), followed by co-culture with tumor HSCs (tHSCs; mDCs to tHSCs ratio, 20: 1) for applied time, the ROS contents and mitochondrial depolarization were tested by H2DCFDA dye (A) and JC-1 dye (B) assays, respectively; Relative expression of listed genes were tested by qPCR assay (C-E). "Ctrl" stands for mDCs only. "PBS" stands for vehicle control PBS. Data are presented as the mean ± standard deviation (n=5). * P < 0.05 vs. "PBS" group. The experiments in this figure were repeated four times, and similar results were obtained. AGING

Figure 5. Nrf2 activation mediates splenic T cell inhibition by tHSCs-stimulated mDCs.
The stable bone marrow-derived dendritic cells (mDCs), with Nrf2 shRNA ("sh-Nrf2") or Nrf2 KO construct ("ko-Nrf2") as well as the parental control mDCs ("Pare") were co-cultured with tumor HSCs (tHSCs) for 24h; Stimulated mDCs were then co-cultured with splenic T cells; OVA-II peptide CTL assay activity (after 24h, A) and LPS (100 ng/mL)-induced IFN-γ production (after 24h, B) were tested. Data are presented as the mean ± standard deviation (n=5). "Ctrl" stands for mDCs only. * P < 0.05 vs. "Ctrl". # P < 0.05 vs. "Pare" group. The experiments in this figure were repeated three times, and similar results were obtained. lead to increased ligation of PD-1 receptor on T-cells. This will lead to T cell apoptosis and suppression of Tcell-mediated tumor cell apoptosis [36][37][38]. Our previous study has shown that tHSCs significantly induced DIgR2 expression in mDCs. The latter is a key negative regulator of DC-initiated T-cell response [12].
ROS will induce Keap1 acetylation, causing Nrf2-Keap1 disassociation and Nrf2 signaling activation [22,39]. One novel finding of the current study is that tHSCs co-culture induced significant ROS production and oxidative stress in mDCs. In tHSCs-treated mDCs ROS production and mitochondrial depolarization were detected. Importantly, ROS production should be the direct cause of Nrf2 activation and DIgR2 expression in mDCs. Two ROS scavengers, NAC and MnTBAP, not only blocked tHSCsinduced ROS production, but also inhibited Nrf2 activation and DIgR2 expression in mDCs. The underlying mechanisms of ROS-mediated Nrf2 activation in tHSCs-treated mDCs need further characterizations.

Rat HCC tumor model
As previously described [43,44], MRH HCC cells [43,44] were initially injected to the flanks of the buffalo rats. Within four weeks the xenograft tumors were established. HCC tumors were cut into small pieces (2×1×1 mm 3 ), transplanted to rat livers [43]. Using a ultrasound method in situ tumor growth was confirmed. The animal protocols were approved by Anhui Medical University's IACUC and Ethics Board.

Primary culture of mDCs and tHSCs-mDCs coculture
As previously described [43,46], the bone marrow was isolated. The red blood cells were abolished, and bone marrow cells were cultured in the described medium [43,44] plus GM-CSF (Sigma) and IL-4 [16,17]. The non-adherent cells were acquired from the proliferating cell clusters, harvested, washed, resuspended and cultured in medium described [46]. For the co-culture experiments, in each well 2.5×10 5 mDCs were incubated with 1.25×10 4 tHSCs (20: 1, mDCs to HSCs). AGING Primary culture of spleen T cells As described previously [43,44], the minced rat spleens were filtered. Splenocytes were isolated from erythrocytes via centrifugation of the cell suspension on a Ficoll gradient (Histopaque 1083, Sigma) [47]. The cells were layered onto the top of the gradient in a 10-ml Falcon tube, followed by centrifugation at 800×g for 20 min at room temperature. Lymphocytes (mainly T-cells) were collected, washed and cultured as described [43,44].

RNA extraction and qPCR
Trizol reagents (Promega, Madison, WI) were utilized for RNA extraction. A SYBR Green PCR kit (Applied Biosystems, Foster City, CA) was utilized for reverse transcription. qPCR assays were performed using the previously described protocol [16,17]. The product melting temperature was calculated via using a melt curve analysis. We utilized the 2 −∆∆Ct method to quantify targeted gene expression, using glyceraldehyde-3phosphatedehydrogenase (GAPDH) as the reference gene and internal control.

Western blotting
Lysates (30 μg proteins of each treatment in each lane) were separated by a 10% SDS-PAGE gel, transferred to a polyvinylidene difluoride (PVDF) blot (Millipore, Temecula, CA) [48]. After incubation in PBST with 10% non-fat milk, the blot was incubated with applied primary and secondary antibodies. To visualize the targeted band, we utilized an enhanced chemiluminescence (ECL, Roche, Shanghai, China) detection kit. An ImageJ software was applied to quantify the band intensity (total gray). The nuclear lysates were isolated by using a previously described protocol [49].

Co-immunoprecipitation (Co-IP) assay
As described [24], following co-culture with tHSC, 500 μg of total cell lysates of mDCs were first pre-cleared by using A/G Sepharose ("Beads", Sigma-Aldrich). Thereafter, an anti-Keap1 antibody (Abcam) was included in the pre-cleared lysates overnight, followed by adding back the protein A/G Sepharose for 2h. Keap1-immunoprecipitated Nrf2 was then tested via Western blotting.

Reactive oxygen species (ROS) detection
H2DCFDA (dichlorofluorescin diacetate), a fluorogenic dye, measures hydroxyl, peroxyl and other ROS activity. In brief, following co-culture with tHSCs, mDCs were incubated with H2DCFDA (5 μM, Abcam) dye for 45 min under dark. H2DCFDA absorbance was detected by a fluorescence spectroscopy with excitation and emission at 495 nm and 529 nm, respectively.

Mitochondrial depolarization assay
Tetraethylbenzimidazolylcarbocyanine iodide (JC-1) is a cationic dye that accumulates in energized mitochondria. It forms green monomers after mitochondrial depolarization. In brief, following co-culture with tHSC, mDCs were incubated with JC-1 dye (10 μM, Abcam) dye for 30 min under dark at the room temperature. At the test wavelength of 530 nm JC-1 green fluorescence absorbance was tested.

Short hairpin RNA (shRNA)-mediated stable knockdown of Nrf2 or Keap1
The Nrf2 lentiviral particles or the Keap1 shRNA lentiviral particles were purchased from Shanghai Genechem (Shanghai, China), added to mDCs for 12h. Cells were then cultured in fresh complete medium (with 10% FBS), following by selection with puromycin (2.0 μg/mL) for another 48h. In the stable cells over 95% of knockdown of target protein (Nrf2 or Keap1) was detected.

Nrf2 knockout
The single-guide RNA (sgRNA) targeting Nrf2 was sub-cloned into a lenti-CRISPR-GFP-puro construct. The Nrf2 knockout (KO) construct was transfected to mDCs by Lipofectamine 2000. mDCs were further subjected to selection with puromycin (2.0 μg/mL) for 48h. In the stable cells Nrf2 KO was verified by Western blotting and qPCR.

Statistical analysis
All statistical analyses were performed by using SPSS 18.0 software. Values are expressed as the mean ± standard deviation (SD). P < 0.05 was considered to indicate a statistically significant result.