Chimeric Vaccine Stimulation of Human Dendritic Cell Indoleamine 2, 3-Dioxygenase Occurs via the Non-Canonical NF-κB Pathway

A chimeric protein vaccine composed of the cholera toxin B subunit fused to proinsulin (CTB-INS) was shown to suppress type 1 diabetes onset in NOD mice and upregulate biosynthesis of the tryptophan catabolic enzyme indoleamine 2, 3-dioxygenase (IDO1) in human dendritic cells (DCs). Here we demonstrate siRNA inhibition of the NF-κB-inducing kinase (NIK) suppresses vaccine-induced IDO1 biosynthesis as well as IKKα phosphorylation. Chromatin immunoprecipitation (ChIP) analysis of CTB-INS inoculated DCs showed that RelB bound to NF-κB consensus sequences in the IDO1 promoter, suggesting vaccine stimulation of the non-canonical NF-κB pathway activates IDO1 expression in vivo. The addition of Tumor Necrosis Factor Associated Factors (TRAF) TRAF 2, 3 and TRAF6 blocking peptides to vaccine inoculated DCs was shown to inhibit IDO1 biosynthesis. This experimental outcome suggests vaccine activation of the TNFR super-family receptor pathway leads to upregulation of IDO1 biosynthesis in CTB-INS inoculated dendritic cells. Together, our experimental data suggest the CTB-INS vaccine uses a TNFR-dependent signaling pathway of the non-canonical NF-κB signaling pathway resulting in suppression of dendritic cell mediated type 1 diabetes autoimmunity.


Introduction
Type 1 diabetes (T1D) is a well-studied prototypic tissue specific autoimmune disease resulting from auto-reactive lymphocyte destruction of the pancreatic islet insulin-producing β-cells [1,2]. The progressive loss of islet β-cell function leads to insulin deficiency and high blood glucose levels (hyperglycemia). Increased levels of cellular oxidative stress and chronic inflammation generated by hyperglycemia leads to neural and circulatory complications that result in an Here we focus on identification of non-canonical NF-κB signaling pathway contributions to CTB-INS vaccine induction of IDO1 in human dendritic cells as a prerequisite for application of chimeric vaccine immune suppression strategies in the clinic.

Materials and Methods
Construction of a bacterial expression vector containing the cholera toxin B subunit-proinsulin gene A DNA sequence encoding 258bp of the human proinsulin gene (INS M12913.1) was linked to the carboxyl-terminus of a DNA fragment (309bp) encoding the cholera toxin B subunit gene (CTB U25679.1) to generate the fusion gene CTB-INS according to a previously used protocol [13] (Fig 1).

Expression and purification of CTB-INS fusion protein in E. coli
The E. coli strain BL21 was transformed with pRSET-CTB-INS as previously described [13].

Ethics
Ex vivo experiments on monocyte-derived DCs were performed, with aphaeresis blood provided by the Life Stream Blood Bank (San Bernardino, CA). These experiments were approved by the Loma Linda University Adventist Health Sciences Center Institutional Review Board and blood donor written consent. Blood donor information was anonymized and de-identified prior to the study Isolation and culture of monocyte-derived dendritic cells from human peripheral blood Monocyte-derived dendritic cells (MoDCs) were prepared from freshly collected human peripheral blood cells isolated from aphaeresis filter cones obtained from the LifeStream blood bank (San Bernardino, CA). The blood was incubated with a red blood cell lysis buffer (3.0 mL Lysis Buffer/ mL of blood) containing 8.3g/L NH 4 Cl, 1g/L KHCO 3 , and 1.8 mL 5% EDTA (Boston Bioproducts), and centrifuged for 5 minutes at 1,500 rpm at 4°C in a Beckman Coulter Allegra X-15R centrifuge, equipped with a SX4750 rotor. After a total of 3 washes in PBS to remove cellular debris and hemoglobin CD14 + monocytes were obtained from the total lymphocyte fraction by incubation with anti-CD14 antibodies bound to magnetic beads for 15 minutes at 4°C (Miltenyi Biotech, Auburn, CA). The monocytes were separated from other immune cells by binding to a magnetic MACS column followed by elution of all other leucocytes (Miltenyi Biotech, Auburn, CA). The monocytes were eluted from the column and cultured at a concentration of 2-9 x 10 6 cells/well in 6-well non-pyrogenic polystyrene culture plates in RPMI 1640 culture medium (Mediatech Inc. Manassas, VA, USA), supplemented with 10% FBS, 1 mM glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, 50 ng/ml human recombinant GMCSF, and 10 ng/ml human recombinant IL-4 (ProSpec-Tany), at 37°C in a humidified atmosphere of 5% CO 2 (Preprotech, Rocky Hill, NJ). The monocyte cell culture was fed at 2-day intervals by gentle replacement of 50% of the medium with fresh pre-warmed culture medium. The cells were cultured for a total of 6 days to allow monocyte differentiation into DCs prior to vaccine treatment. The cells were monitored by phase contrast microscopy to assess dendrite formation, a marker indicating DC differentiation.

Small interfering RNA (siRNA) transfection
No pharmacological inhibitors for IKKα exist that selectively block the non-canonical pathway of NF-κB activation [14,23]. Here we used siRNA to specifically target this pathway. To define the regulatory effect of the NF-κB pathway on CTB-INS-induced IDO expression, human NIK-small interfering RNA (NIK siRNA, sc-36065) and non-targeting siRNA (Control siRNA Fluorescein conjugate, sc-36869) were purchased from Santa Cruz Biotechnology (Santa Cruz, Delaware, CA, USA). Monocytes were cultured in six-well plates for 4 days with 50 ng/ml of hGM-CSF and 10 ng/ml of IL-4 for differentiate the mature DCs. siRNAs were transfected into DC cells using Lipofectamine1 RNAiMAX reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's protocol. Three microliters of 10 μM siRNA was mixture in 150 μl of Opti-MEN (Gibco-Life Technologies, Paisley,UK), while 9 μl of Lipofectamine1 RNAiMAX reagent was incubated in 150 μl of Opti-MEN at room temperature for 5 min. Then the diluted siRNA and Lipofectamine1 RNAiMAX reagent were incubated for a further 20 min at room temperature for complex formation. The complexes were added to wells. The final siRNA concentration was 25 pmol. DC continued to be incubated at 37°C in 5% humidified CO 2 for 48 h which was sufficient to significantly knock down the target protein levels. Expression of IDO1 was induced by 5 μg of CTB-INS for 24h after siRNA transfection. To evaluate transfection efficiency, FITC-labeled control RNA was substituted for siRNA. After 24 hours, incubation, the transfected DCs were analyzed by fluorescence microscopy for intracellular FITC content. To confirm IKKα phosphorylation, DCs were treated with 10μg/ml of CTB-INS for 6hrs after transfection and lysed in 100 μL buffer C/well containing phosphatase inhibitors (50 mM Sodium-beta-glycerophosphate, 1mM Sodium fluoride, 1 mM Sodium-ortho-vanadate). Western blot analysis with Anti-IKK alpha (phospho S176+S180) (Abcam 1:1000) and anti-rabbit IgG whole molecule conjugated AP (Sigma-Aldrich 1:1000) was performed prior to band detection on x-ray film (Kodak X-Omat) of IKKα.expression.

Total RNA preparation and reverse transcription polymerase chain reaction (RT-PCR)
Total RNA from 1 X 10 6 DC was prepared using Trizol (Invitrogen, Carlsbad, CA, USA) and complementary DNA was synthesized from 2 μg total RNA with oligo (dT) primer in a 20 μl reaction volumn according to the manufacturer's recommendations (Thermo Fisher Scientific Inc, Waltham, MA, USA). Polymerase chain reaction (PCR) amplification was performed at 95°C for 1m, 58°C for 1m, 72°C for 30s, and PCR was done for 35 cycles. The primers used in this study were NIK (h)-PR (sc-36065-PR, Santa Cruze), product size 537 bp; and β-actin forward, 5'-GCA TTG CTT TCG TGT AAA TTA TGT-3' β-actin reverse, 5'-ACC AAA AGC CTT CAT ACA TCT CA-3', product size 211 bps. The PCR products were size-separated on 1.5% agarose gels and visualized by Et-Br DNA gel staining.

ChIP analysis of CTB-INS induction of NF-κB activation in vivo
A chromatin immunoprecipitation (ChIP) assay was performed to identify the specific binding sequences in the IDO1 promoter region for the non-canonical NF-κB subunits RelB protein using a MAGnify TM Chromatin Immunoprecipitation System (Invitrogen) according to the manufacturer's instructions. 3-8 x 10 6 Human dendritic cells were left unstimulated or were stimulated with CTB-INS for 3h, after which DC cells were harvested and washed with 1XPBS and were fixed in formaldehyde (Sigma, St Louis, MO, USA) to a final concentration of 1%. After 10 min, 1.25M glycine was added to stop crosslinking reaction. After centrifugation, cells were lysed for 5 min in Lysis buffer supplemented with protease inhibitors. Chromatin was sheared by sonication (5 X 12s at one-fifth of the maximum potency) with a Sonic 60 Dismembrator (Fisher Scientific, Sunnyvale, CA, USA), centrifuged to pellet debris, and diluted in Dilution Buffer which was recommend in manufacturer's instructions. Fragmented chromatin was immunopreciated with a ChIP-grade antibody against RelB (GeneTex, Irvine, CA, USA) which was coupled with Dynabeads 1, at 4°C overnight. Immune complexes were washed with IP Buffer 1 and 2 in the DynaMag TM -PCR Magnet. For reversing the crosslinking, Reverse Crosslinking Buffer with proteinase K was added both input control (fragmented chromatin without immunoprecipitation) and immune complexes and incubate at 55°C for 15 min. The DNA was purified with DNA Purification Magnetic Beads and buffers provided in the kit according to the manufacturer's instructions. The immunoprecipitated DNA was used in each real-time PCR assay using primers specific for indicated regions of the DNA. The primers were designed using Primer Express 2. The cycling conditions were set as follows: an initial denaturation step of 95°C for 10 min to activate the iTaq DNA polymerase, followed by 40 cycles of denaturation at 95°C for 15 s, and annealing at 60°C for 1 min. The amplification process was followed by a melting curve analysis, ranging from 65°C to 95°C, with temperature increasing at steps of 2°C every 1 min. Baseline and threshold cycles (Ct), were automatically determined using the Bio-Rad CFX Manager 2.1. The samples were electrophoresed on a 1.5% (w/v) agarose gel, and the banding pattern observed under UV light. Two biological replicates for each sample were used for real-time PCR analysis and three technical replicates were analyzed for each biological replicate.

Blocking TNFR activation of IDO1 biosynthesis
Peptides containing the CD40 receptor TRAF2, 3 and TRAF6 binding sites were linked to the TAT 47-57 cell penetrating peptide. The sequences for the CD40-TRAF2, 3 and the CD40-TRAF6 blocking peptides were NH 2 -NTAAPVQETLHGYGRKKRRQRRR-OH and NH 2 -KQEPQEIDFPDD YGRKKRRQRRR-OH respectively. The TAT 47-57 sequence is underlined. Control peptides consisted of either TAT 47-57 alone or TAT 47-57 linked to a scrambled peptide. The peptides were manufactured by Proteintech Group (San Diego, CA) and were low in endotoxin and > 98% pure as measured by HPLC [24,25].

IDO1 expression following CTB-INS incubation
Concentrations of 0.1-10 μg/ml CTB-INS were used to assess the minimal concentration of CTB-INS needed to allow detection of IDO1 in monocyte-derived DCs. Expression of IDO1 induced by CTB-INS occurred at concentrations as low as 0.5μg/ml of CTB-INS (Fig 2A) Monocyte-derived DCs were incubated with CTB-INS for 6 to 96 hours with the medium replaced at 2 day intervals. The levels of IDO1 in vaccine inoculated DCs increased continuously for 96 hours following vaccination (Fig 2B).

CTB-INS stimulation of the non-canonical NF-κB pathway induces IDO1 synthesis in DCs
To assess non-canonical NF-κB pathway contributions to CTB-INS-induced IDO1 expression we used siRNA technology to knock down the non-canonical pathway-dependent kinase NIK and the level of IDO1 expression in treated DCs measured. The knockdown of NIK in CTB-INS stimulated DCs resulted in a significant reduction in NIK mRNA levels ( Fig 3A) and IDO1 protein expression as well as a decrease in phosphorylated IKKα in comparison with non-specific siRNA-treated dendritic cells (Fig 3B). This experimental result demonstrated that CTB-INS-induced IDO expression in human DCs was dependent upon vaccine activation of the non-canonical NF-κB pathway.

CTB-INS leads to non-canonical NF-κB RelB translocation to drive IDO1 expression in DCs in vivo
The IDO1 promoter region was shown to contain three partial RelB/p52 binding sites (AGGA GACACA, GGGAGACAGA, and AGGAGAAAGA), with a consensus noncanonical binding sequence PuGGAGApyTTPu located close to position -2000 (Fig 4A), [21,28,29]. To demonstrate direct binding of RelB/p52 to the IDO1 promoter, we performed a ChIP analysis experiment using RelB binding to pull-down the IDO1 promoter (Fig 4B). Immunoprecipitation of RelB induced by CTB-INS showed binding to all three IDO1 non-canonical binding sequences with increased binding at the GGGAGACAGA promoter sequence (Fig 4B).

Vaccine stimulation of the TNFR signaling pathway induces IDO1 biosynthesis in human DCs
Control of NIK post-translational stability may be essential for non-canonical NF-κB signaling modulation. Therefore, the control of NIK stability is one of the prime questions for understanding regulation of the non-canonical NF-κB signaling pathway. Mounting evidence suggests that TNF receptor-associated factors, TRAF2, TRAF3 and TRAF 6, are critical molecules involved in negative regulation of NIK activity [30][31][32]. (Fig 5) Pharmacological inactivation of these proteins or their deletion also allows for basal NIK accumulation in the absence of ligand [15,17,19,24,25,33,34].
The engagement of CD40 by CD40L promotes clustering of CD40 inducing the recruitment of adapter proteins known as TNFR-associated factors (TRAFs) to the cytoplasmic domain of CD40 [33]. Previous reports showed that cell permeable peptides that include the TRAF2, 3 or TRAF6 binding site to CD40 are able to block the CD40-TRAF signaling pathway [24,32,33,[35][36][37][38]. Inhibition of the TNFR pathway permits examination of its role in induction of IDO1 in vaccinated dendritic cells. Therefore, monocyte-derived DCs were incubated with peptides containing the amino acid sequence of the TRAF2, 3 and the TRAF6 binding sites to CD40. The DCs were then stimulated with CD40 ligand (CD154), and CTB-INS. The CD40-TRAF2, 3 and CD40-TRAF6 blocking peptides were shown to impair upregulation of IDO1 in response to CD154, and CTB-INS treatment. The most impairment Activation of the non-canonical NF-κB pathway. In the basal inactive state (left), the TRAF-cIAP complex catalyzes ubiquitination of NIK, leading to constitutive NIK degradation in the proteasome leaving p100-containing RelB complexes isolated in the cytoplasm. During activation (right), the TRAF-cIAP complex is recruited to the CD40 receptor. Upon ligand binding, TRAF2-mediated, ubiquitination of cIAP1/2 switches its ubiquitin ligase activity from NIK to TRAF3. The resultant TRAF3 degradation destabilizes the TRAF-cIAP complex allowing accumulation of newly synthesized NIK. Phosphorylated NIK then transfers a phosphate to IKKα. Now activated, IKKα phosphorylates p100 leading to its partial proteosomal degradation releasing p52: RelB heterodimers that translocate into the nucleus. Further, activated IKKα phosphorylates NIK, destabilizing it thereby limiting downstream activation events.

CTB-INS as a ligand for members of the TNFR Superfamily
Members of the Tumor Necrosis Factor (TNF) receptor family have been shown to stimulate DC maturation or modulate peripheral tolerance in autoimmunity by upregulation of IDO1 [14,22,39]. Both CTB and LTB enterotoxin protein binding subunits were shown to stimulate antigen presenting cell CD40 surface expression and DCs were found to upregulate IDO1 mediated immune suppression through activation of the NF-κB non-canonical signaling pathway [14]. Protein functional homology analysis (PROPSEARCH TM ) identified the probability of functional homology between CTB-INS and the TNF subfamily of ligands to be >87%, (unpublished data). Based on the data in (Fig 6) we hypothesize that CTB-INS interacts with TNF receptors to stimulate IDO1 synthesis in vaccinated DCs. To test this hypothesis, we aligned CTB-INS protein amino acid sequence with the following tumor necrosis factor (TNF) superfamily member ligands: CD40L (Accession: NP_000065.1), TNFR14L (Accession: NP_003798.2), RANKL (Accession: NP_003692.1), and BAFF (Accession: NP_006564.1) using the T-Coffee server. We observed that the CTB-INS vaccine shares significant levels of amino sequence homology with ligands of the TNFR superfamily (Fig 7).

Discussion
The mechanism of CTB-INS induction IDO1 biosynthesis was shown to be dependent on the NF-κB signaling pathway [13]. However, the relative contributions of canonical and noncanonical branches of this pathway to IDO1 up regulation remain unknown. Earlier work by Tas and his colleagues showed that CD40L was responsible for stimulation of IDO1 via the non-canonical pathway [14], suggesting this pathway could play a significant role in CTB-INS induction of IDO1 in human DCs. Based on NIK-dependent activation experiments, our data suggest CTB-INS induces IDO1 in human monocyte-derived DCs via the non-canonical NF- κB pathway. In addition, ChIP analysis experiments showed that NF-κB RelB-p52 dimers bound to defined consensus sequences within the IDO1 promoter in vivo, suggesting the noncanonical signaling pathway is active in vaccine induction of IDO1 in human DCs. Blocking TRAF adaptor molecule functions was shown to inhibit IDO1 biosynthesis in vaccinated DCs suggesting upregulation of IDO may occur through TNF receptor family stimulation of the NF-κB non-canonical signal transduction pathway [31]. In the TNF-activated signal transduction pathway, NIK is known to interact with TRAF2, and TRAF3 leading to non-canonical NF-κB activation [32].
Induction of IDO1 depends on fusion of CTB to proinsulin [13], suggesting that the vaccine may bind as a ligand to receptors responsible for NF-κB non-canonical pathway activation of IDO1 expression. Several ligands of the Tumor Necrosis Family Receptor (TNFR) superfamily were shown to activate the non-canonical NF-κB pathway [14,[40][41][42][43][44]. For suggestive evidence of a link between CTB-INS with TNFR, we compared amino acid sequences of CTB-INS with those of four ligands of the TNFR family to assess any type of functional homology. We found that several areas where the vaccine could act as a TNFR ligand, represented by areas of greater amino acid homology (Fig 7).
The association between the ligand and its potential receptor suggests that CTB-INS receptor binding may involve specificity of the autoantigen for its receptor rather than existing as a general mechanism for binding all CTB-autoantigen conjugates. Our experimental data suggests that CTB-INS induces non-canonical NF-κB signaling which is driven by TRAFs as TNFR signal mediators. Although, there is evidence that CTB-INS elicits immunosuppressive effects through TNFRs, further experiments are needed to determine the probability of CTB-INS binding to individual members of the TNFR family.
The B-cell activating factor (BAFF) predominantly expressed in B cells, differs from many other TNFR superfamily members in that it generally activates the non-canonical NF-κB signaling pathway with only weak induction of canonical NF-κB pathway signaling [41,45]. This unique feature of the BAFF receptor (BAFFR) is due primarily to its possession of an atypical TRAF-binding sequence, which interacts with TRAF3 but not with TRAF2 [41]. The BAFFRmediated induction of p100 processing to p52 contributes to the survival of transitional and mature B cells, likely through induction of anti-apoptotic genes like bcl-2 and bcl-x [41,45].
The CD40 molecule is a TNFR member expressed on a variety of cell types, including B cells, dendritic cells, monocytes, endothelial epithelial cells, and neurons [39,46]. Activated T cells primarily express the ligand of CD40, alternatively referred to as CD40L or (CD154). In the immune system, a major function of CD40 signaling is to regulate B-cell activation and differentiation events, including proliferation and survival of activated B cells, germinal center formation, and antibody isotype switching. Another major function of CD40 is to mediate dendritic cell maturation and antigen presentation. Unlike BAFFR, CD40 elicits strong signals that target both the canonical and non-canonical NF-κB pathways [14,39]. Upon ligation by CD40L, CD40 interacts via two different TRAF-binding motifs that include TRAF1, 2, 3, 5, and 6, and this interaction leads to proteolysis of both TRAF2 and TRAF3 [43,47]. As indicated above, the degradation of TRAF2 and TRAF3 represent an important step in the activation of the non-canonical NF-κB signaling pathway [15,16,34].
The herpesvirus entry mediator (HVEM) or tumor necrosis factor receptor superfamily member 14 (TNFRSF-14) is a protein originally known as herpesvirus entry mediator A (HveA). Both HveB and HveC are structurally unrelated proteins of the immunoglobulin superfamily [48]. HvA is also known as Cluster of Differentiation CD270 [40]. Moreover it is also referred to as ATAR (another TRAF-associated receptor). Interactions between TNFRSF-14 and TRAF2 were shown to activate the non-canonical NF-κB signaling pathway [40].
The Receptor Activator of Nuclear Factor κ B (RANK) is best known for its role in osteoclastogenesis [15,44]. However, it also regulates important immune functions that include dendritic cell survival and lymphoid organogenesis [42]. RANK is expressed on osteoclast precursors, dendritic cells, and activated B cells, and in general, RANK signaling was shown to promote cell survival and differentiation. Analogous with CD40, the cytoplasmic domain of RANK was shown to bind TRAF1, 2, 3, 5, and 6 and mediates activation of both canonical and non-canonical NF-κB signaling pathways. Genetic evidence suggests an essential role for RANK-stimulated activation of non-canonical NF-κB activation during osteoclastogenesis and bone metabolism [44]. The non-canonical NF-κB has been closely linked to immune suppression [13,49]. Several ligands such as Glucocorticoid-induced tumor necrosis factor receptor (GITR) on T cells and its natural ligand, GITRL, on accessory cells contribute to the control of immune homeostasis. Grohmann et al. showed that reverse signaling through GITRL after engagement by soluble GITR initiates the immunoregulatory pathway of tryptophan catabolism in mouse plasmacytoid dendritic cells, by means of noncanonical NF-κB-dependent induction of IDO1 [50]. Additionally, CpG-rich oligodeoxynucleotides activate the immune system, leading to innate and adaptive immune responses that have been shown to promote tolerogenic responses in mouse plasmacytoid dendritic cells in vivo and in an in vitro human DC model. Unveiling a previously undescribed role for TRIF and TRAF6 proteins in Toll-like receptor 9 (TLR9) signaling, it was demonstrated that physical association of TLR9, TRIF and TRAF6 leads to activation of non-canonical NF-κB signaling and the induction of IRF3-and TGF-β-dependent immune-suppressive tryptophan catabolism [51].
Understanding the link between vaccine activation of TNF receptor family members and the activation of non-canonical NF-κB signaling is an important step in elucidation of the mechanism underlying chimeric vaccine induction of immunological tolerance in dendritic cells. Understanding the mechanism of chimeric vaccine modulation of IDO1 induction and suppression of in human dendritic cell activation will facilitate development of chimeric vaccine strategies for effective and safe therapy for type 1 diabetes and a wide range of tissue specific autoimmune diseases.