Signaling through NOD-2 and TLR-4 Bolsters the T cell Priming Capability of Dendritic cells by Inducing Autophagy

T cells play a cardinal role in mediating protection against intracellular pathogens like Mycobacterium tuberculosis (Mtb). It is important to understand the factors that govern the T cell response; thereby can modulate its activity. Dendritic cells (DCs) are the major player in initiation and augmentation of T cell response. Targeting DCs to induce their optimum maturation and activation can lead to a better T cell response. Interestingly, we observed that combinatorial signaling of DCs through NOD-2 and TLR-4 fortified better yield of IL-12p40/70, IL-6 and IFN-γ and upregulated the expression of CD40, CD80 and CD86 costimulatory molecules. Further, we noticed improved phagocytic capabilities of DCs. Furthermore, NOD-2 and TLR-4 induced autophagy in DCs, which enhanced the activation of T cells. This study signifies that NOD-2 and TLR-4 exhibit synergism in invigorating the activity of DCs. Consequently, this strategy may have significant immunotherapeutic potential in bolstering the function of DCs and thus improving the immunity against pathogens.

Scientific RepoRts | 6:19084 | DOI: 10.1038/srep19084 Results N 2 T 4 stimulation of DCs enhances cytokine releases. Initiation of immune response is critically dependent on the activation of DCs. This process starts with the release of cytokines. We observed that DCs triggered through TLR-4 showed dose dependent increase in the release of IL-6 ( Fig. S1). However, NOD-2 triggering showed remarkably lesser production of IL-6, compared to TLR-4. Interestingly, combinatorial signaling through TLR-4 and NOD-2 (N 2 T 4 ) exhibited synergistic impact and significantly enhanced the yield of IL-6 (p < 0.0001), IL-12p40/70 (p < 0.0001) and IFN-γ (p < 0.0001) compared to controls (N 2 L or T 4 L) ( Fig. 1A-C). This observation related to IL-6, IL-12 and IFN-γ was further substantiated at mRNA level by RT-qPCR ( Fig. 1D-F). Noteworthy, optimum release of IL-6 was observed at 10 μ g/ml of N 2 L and 5 ng/ml concentration of T 4 L (Fig. S1A, B). Therefore, these doses were selected for all the experiments. To rule out the possibility of any contaminating cells in the results, DCs isolated by MACS showed 95% purity. These DCs were triggered through N 2 T 4 and release of IL-12p40/70 was estimated in the culture SNs ( Fig. S2A-D). We observed the similar pattern in the production of IL-12p40/70, as was noticed with cultured DCs (Fig. S2D, Fig. 1B).

Signaling delivered through N 2 T 4 induces maturation and activation of DCs. Maturation of DCs
involves upregulation of expression of MHC-II and costimulatory molecules 14 . Intriguingly, we noted that signaling delivered through N 2 T 4 augmented the expression of CD40 (p < 0.01), CD86 (p < 0.05), CD80 (p < 0.05) and MHC-II (p < 0.01), when compared to untreated DCs (uDCs) or treated with N 2 L or T 4 L ( Fig. 2A-D). Similar results were noted with MACS purified DCs (Fig. S2E, F).
Next, we studied the potential of N 2 T 4 triggered DCs to activate T cells. N 2 T 4 activated DCs were co-cultured with anti-CD3 stimulated CD4 T cells. Interestingly, we observed that N 2 T 4 stimulated DCs induced significant (p < 0.001) increase in the proliferation of T cells (Fig. 2E). Further, it was noticed that T cells cocultured with activated DCs showed better production of IFN-γ (p < 0.05) than control cells (Fig. 2F). These data suggest that combinatorial signaling delivered through N 2 T 4 showed synergism in activating T cells. N 2 T 4 signaling substantially improved the phagocytosis competence of DCs. DCs is reasonably critical in capturing antigens. We observed noteworthy increase in the antigen uptake by N 2 T 4 triggered DCs than controls. This was evidenced by higher antigen uptake by confocal microscopy (Fig. 3A, B). Further, these results were corroborated by flowcytometry data by significant (p < 0.0001) increase in the dextran-FITC uptake (Fig. 3C). These experiments suggest that N 2 T 4 stimulation of DCs exhibited remarkable synergism between both the molecules in bolstering the antigen uptake by DCs. The 'x axis' signifies concentration of TLR-4L (5 ng/ml) and NOD-2L (10 μ g/ml). Graphs depict mRNA expression of (D) IL-6; (E) IL-12p40; (F) IFN-γ relative to untreated controls by RT-qPCR. Data shown as mean ± SD are representative of two independent experiments. *p < 0.05, ***p < 0.001, ****p < 0.0001.
Scientific RepoRts | 6:19084 | DOI: 10.1038/srep19084 N 2 T 4 stimulation induces autophagy in DCs. Autophagy mediates the intracellular killing of bacteria by targeting antigen to lysosomal degradation pathway 15 . Further, it also contributes in the presentation of antigens via MHC class I and II pathways. We observed that N 2 T 4 stimulation of DCs showed the increment in the conversion of LC3I to LC3II, which are markers for autophagy (Fig. 4A). Further, N 2 T 4 L stimulation showed higher accumulation of LC3II in the presence of bafilomycin, which blocked the fusion of autophagosome with lysosome and thereby preventing the degradation of LC3II (Fig. 4B). Furthermore, we corroborated these results by demonstrating puncta formation through immunofluorescence staining (Fig. 4C,D). N 2 T 4 activated DCs acquired enhanced capability to prime naive T cell. To confirm the potency of N 2 T 4 activated DCs to prime naive T cells, Mtb infected DCs triggered via N 2 T 4 were adoptively transferred into mice. After 5d, it was ascertained that CD4 T cells or CD8 T cells primed by N 2 T 4 activated DCs, efficiently triggered the IFN-γ (p < 0.001) release, as compared to infected or untreated DCs after in vitro stimulation with PPD ( Fig. 5A,B, Fig. S3A,C). The lymphocytes isolated from the mice that were adoptively transferred with infected DCs were cultured in vitro with PPD.
Earlier, we have demonstrated that N 2 T 4 activated DCs showed augmented autophagy. Autophagy is a bactericidal phenomenon but simultaneously it is known to enhance T cell response 15,16 . Therefore, we were curious to study whether N 2 T 4 induced autophagy in DCs exhibits any effect on priming of naive T cells. To confirm this, DCs were incubated with wortmannin to block autophagy, prior to Mtb infection followed by stimulation through N 2 T 4 . We observed that such DCs showed significant (p < 0.001) decline in the secretion of IFN-γ by CD4 T and CD8 T cells (Fig. 5A,B, Fig. S3A-D). We also used another autophagy inhibitor 3MA, to further validate our results (Fig. S3E,F). It is important to mention that the effect of wortmannin used as an autophagy inhibitor showed no effect on the viability of DCs or in the level of costimulatory molecules CD40 and CD86   expressed on DCs ( Fig. S4A-C). Further, we observed significant (p < 0.001) decrease in the proliferation of lymphocytes on in vitro challenge with PPD (Fig. 5C,D). The antigen specificity was proved by in vitro stimulation of lymphocytes isolated from the mice adoptively transferred with Mtb infected DCs treated with N 2 T 4 L with OVA, a non Mtb antigen. Overall the results demonstrate that N 2 T 4 induced autophagy enhances the DCs capacity to activate T cells.

Discussion
Dendritic cells are the major player in the generation of effective T cell responses 17 . Importantly, DCs efficacy depends on many variables, especially maturation status and efficient antigen presentation to naive T cells. However, signaling of DCs through surface receptors in particular, improve their potency in enhancing adaptive immunity 18,19 . Ligation of CD40 on DCs triggers the production of IL-12 and augments their T cell stimulatory capacity 20 . Further, it has been documented that signaling via TLR-4 amplifies the outcome of CD40 response. Cumulative signaling of CD40 and TLR-4 enhances the production of IL-12 by DCs and improve their anti-tumor efficacy 21 . These DCs stimulated T cells against tumor-associated antigens. It suggests that crosstalk between NOD-2 and TLR-4 stimuli may lead to a better performance of DCs; as has been noticed in the case of better release of cytokines, upregulation of costimulatory molecules and phagocytic activity. However, the role of such DCs in the activation of T cells has not yet been studied. Therefore, we thought that cumulative signaling through N 2 T 4 may be imperative in bolstering DCs functions to improve T cells response. We selected N-glycolyl MDP as NOD-2 agonist; since it exhibits 10-100 fold more potent immunogenicity than the commonly studied N-acetylated MDP 22 . We used LPS as a source of TLR-4 ligand. Recently, Food and Drug Administration (FDA) has approved its use in future medicines, which has opened new avenues to harness its remedial potential 23 .
In the current study, delivering combinatorial signals through N 2 T 4 to DCs led to the emergence of following interesting findings: i) enhanced activation and maturation of DCs; ii) augmented phagocytosis by DCs; iii) increased autophagy; iv) improved capability of DCs to prime naive T cells. Intriguingly, we observed that signaling through N 2 T 4 induced robust release of IL-6, IL-12p40/70, and IFN-γ by DCs. Importantly, these cytokines play important role in the activation of not only naive T cells but also helps in stimulating other cells responsible for sustaining immunity, including DCs. Further, IL-12 promotes the differentiation of naive CD4 T cells to Th1 subtype. Th1 cells perform cardinal function to protect against intracellular pathogens like Mtb 24 .

IFN-γ is well known cytokine for the induction of the expression of MHC-I and MHC-II molecules.
It is well established that the expression of costimulatory molecules is critical for the activation of T cells 18,19,25,26 . Interestingly, we also observed the upregulation of costimulatory molecules such as CD40, CD80 and CD86 on N 2 T 4 activated DCs. Optimum expression of costimulatory molecules on DCs is exceedingly essential in deciding the activation or anergy of naive T cells 27 .
To prime T cells, the primary function of the immature DC is to capture antigen. Importantly, N 2 T 4 triggered DCs displayed better phagocytic capacity. After antigen is captured, it is processed by exogenous and endogenous pathways 28 . In addition to classical pathways, autophagy has also been reported to enhance the antigen presentation by APCs to CD4 and CD8 T cells 29,30 . It is worth to mention here, that N 2 T 4 stimulation of DCs augments autophagy. Autophagy plays a critical role in elimination of pathogens by targeting them to lysosomal degradation pathway 31 . Importantly, we observed that blocking of autophagy induced via N 2 T 4 signaling in DCs suppresses IFN-γ release by T cells. Thus this study opens new avenue of exploring immunomodulators to invigorate the potency of DCs to prime naive T cells.

Culture of bone marrow derived DCs and their stimulation through NOD-2 and TLR-4 (N 2 T 4 ).
Bone marrow derived DCs were cultured according to Lutz et al. 32 . Briefly, bone marrow cells (BMCs) were flushed aseptically from femurs and tibia. For DC cultures, cells were grown in RPMI 1640 (Invitrogen, Life Technologies, Eugene, OR) containing FCS-10% (GIBCO, Grand Island, NY) supplemented with penicillin (100 U/ml), streptomycin (100 mg/ml), and L-glutamine (100 mM), and granulocyte-macrophage colony-stimulating factor (GMCSF) (2 ng/ml) and murine rIL-4 (4 ng/ml) for 6d. Cultures were maintained in a humidified atmosphere, CO 2 (5%) at 37 °C. The medium was replenished on 3d. Later, DCs were harvested, washed and stimulated for 24 h with N-glycolyl MDP (10 μ g/ml) and LPS (5 ng/ml) as ligands of NOD-2 (N 2 L) and TLR-4 (T 4 L), respectively. These doses were selected on the basis of optimum secretion of IL-6 observed during checker board titration of the doses of NOD-2 and TLR-4 (Fig. S1). Bone marrow derived DCs were purified through magnetic associated cell sorting (MACS) as per the manufacturer's instructions (BD Biosciences, San Diego, CA). Later, purified DCs (95%) were stimulated through N 2 T 4 , as described above.  Antigen uptake. DCs were stimulated through N 2 T 4 and controls with N 2 and T 4 for 24 h. Later, activated DCs were incubated with dextran-FITC (1 mg/ml) for 2 h. It was followed by extensive washing with PBS. The cultures were fixed with paraformaldehyde and confocal microscopy (NIKON A1) was performed.
Western Blotting. DCs were stimulated either through N 2 T 4 or controls via N 2 or T 4 in the presence or absence of bafilomycin (100 nM) or 2 h. Later, cells were harvested, washed, and lysed in lysis buffer (RIPA buffer, protease and phosphatase inhibitor cocktail). In SNs, proteins were estimated and equal concentration was subjected to SDS-PAGE. After transfer to nitrocellulose membrane and subsequent blocking, the membranes were immunoblotted with Abs against LC3-I/ LC3II and actin as a loading control. Blots were developed using chemiluminescence kit (Amersham Pharmacia Biotech, Buckinghamshire, UK). Blots were scanned with the help of phosphoimager (Fujifilm, Tokyo, Japan) and image analysis was performed with MultiGuage software.
Immunofluorescence staining. DCs were stimulated through N 2 T 4 and controls via N 2 or T 4 for 4 h. Later, cells were harvested and fixed with 4X paraformaldehyde for 10-15 mins. It was followed by treatment with tween-20 (0.1%) for 15 sec. Cells were extensively washed with PBS. To block non-specific sites, DCs were incubated with BSA (5%) for 3 h, followed by rabbit anti-mouse LC3 Ab for 4 h. After 3X washing, cells were incubated with anti-rabbit FITC for 1 h. Cells were imaged under fluorescence microscopy.
RT-qPCR for the quantification of IFN-γ, IL-6 and IL-12p40. Total RNA was isolated by trizol reagent from DCs stimulated through N 2 T 4 or controls via N 2 or T 4 for 6 h, according to the manufacturer's instructions (Invitrogen, Carisbad, CA). RNA was quantified with the help of NanoDrop spectrophotometer. A260/A280 ratio of all samples was in the range of 1.90 to 2.00. Intactness of RNA samples was determined with the help of formaldehyde denaturing agarose gel-electrophoresis. DNA contamination from RNA samples was removed by amplification grade DNase. Briefly, RNA samples (1 μ g) were incubated with DNase (1U) for 15 min in the reaction buffer. After the incubation, DNase was terminated by stop solution. Further, the samples were heated to 70˚C/10 min to inactivate DNase activity. Results are represented in the form of re-expression (fold) relative to untreated controls. Analysis was done by comparative Ct method, whereas Ct values were normalized against house-keeping control actin. Using the comparative Ct method relative gene expression was calculated as 2 (-∆∆Ct) , where ∆Ct = Ct (gene of interest)-Ct (normalizer = β -actin) and the ∆∆Ct = ∆Ct (sample)-∆Ct (calibrator). Calibrator was total RNA from lungs of placebo. RT-qPCR and data analysis was done by Realplex Master cycler (Eppendorf, Hamburg, Germany).
Statistical analysis. Data were examined by one way analysis of variance (ANOVA) with post Tukey-Kramer multiple comparisons test by using Graph Pad Prism software.