Triggering through NOD-2 Differentiates Bone Marrow Precursors to Dendritic Cells with Potent Bactericidal activity

Dendritic cells (DCs) play a crucial role in bridging innate and adaptive immunity by activating naïve T cells. The role of pattern recognition receptors like Toll-Like Receptors and Nod-Like Receptors expressed on DCs is well-defined in the recognition of the pathogens. However, nothing is precisely studied regarding the impact of NOD-2 signaling during the differentiation of DCs. Consequently, we explored the role of NOD-2 signaling in the differentiation of DCs and therefore their capability to activate innate and adaptive immunity. Intriguingly, we observed that NOD-2 stimulated DCs (nDCs) acquired highly activated and matured phenotype and exhibited substantially greater bactericidal activity by robust production of nitric oxide. The mechanism involved in improving the functionality of nDCs was dependent on IFN-αβ signaling, leading to the activation of STAT pathways. Furthermore, we also observed that STAT-1 and STAT-4 dependent maturation and activation of DCs was under the feedback mechanism of SOCS-1 and SOCS-3 proteins. nDCs acquired enhanced potential to activate chiefly Th1 and Th17 immunity. Taken together, these results suggest that nDCs can be exploited as an immunotherapeutic agent in bolstering host immunity and imparting protection against the pathogens.

differentiation of Th17 cells against bacterial insult 12 . TLR-7/TLR-8 agonists impair the differentiation and maturation of DCs 13 . Further, LPS blocks the differentiation of monocytes to DCs in vivo 14 . In addition, other pattern recognition receptors (PRRs) like Nucleotide-binding oligomerization domain like receptors or NOD-like receptors (NLRs) are known to play strategic role in regulating DCs activity. Recently, NOD-2 which is an important member of NLRs family has been acknowledged in regulating the DCs activity with better efficiency to present antigen to CD8 T cells 15 . However, nothing has been documented about the role of NOD-2 signaling in the differentiation of DCs. NOD-2 consists of an N-terminal caspase recruitment domain (CARD), an intermediate NOD and a C-terminal leucine-rich repeats (LRRs) domain. It recognizes the peptidoglycan derivatives muramyl dipeptide (MDP). N-glycolyl muramyl dipeptide (NGMDP) is a modified agonist of NOD-2 with significantly better stimulatory activity than MDP 16,17 . It is important to mention that NOD-2 is expressed in the cytoplasm and to ligate with receptors, a ligand for NOD-2 is internalized into acidified vesicles by endocytosis dependent on the clathrin and dyamin pathways 18,19 . Here, we explore the influence of signaling of NOD-2 with NGMDP in the activation and differentiation of DCs with improved functions. We observed that nDCs exhibit activated phenotype with high expression of costimulatory molecules, abundant release of cytokines and NO. Importantly, nDCs were highly efficient in restricting the intracellular growth of Mtb. This study for the first time unveils the role of NOD-2 in the differentiation of the DCs, which make their contribution more specialized towards innate immunity.

Results
Signaling through NOD-2 influences the differentiation of DCs. Bone marrow cells (BMCs) were cultured in the presence of GMCSF + IL-4 along with NGMDP, a ligand for NOD-2 (NOD-2L). Triggering through innate ligands blocks the differentiation of BMCs to DCs. Therefore, we first studied the expression of CD11c, which is a classical marker of DCs. We observed that DCs cultured with NOD-2L (nDCs) showed a similar expression of CD11c, as control DCs (DCs differentiated in the presence of GMCSF + IL-4) (Fig. 1A). However, these cells showed enlargement in the size, as demonstrated by scanning electron microscopy (Fig. 1B). We also noted a significant (p < 0.01) expansion in the yield of nDCs (Fig. 1C). This information was corroborated by bright field microscopy ( Fig. 1D). Furthermore, nDCs exhibited substantial enhancement in the release of IL-6 (p < 0.01) and IL-12 (p < 0.001) than control DCs (cDCs) (Fig. 1E). All the observed changes in the nDCs were noticed in a dose dependent fashion ( Fig. 1A-E). Since optimum changes in the functionality (release of IL-6 and IL-12) of nDCs were observed at 6 μ g/ml of NGMDP, hence this dose was used in all subsequent experiments.
DCs acquired mature phenotype on signaling through NOD-2. Efficiency of DCs depends preferably on three important factors: i) the expression of MHC and costimulatory molecules and release of cytokines; (ii) migration from periphery to the T cell areas of lymphoid organs to prime T cells; (iii) antigen uptake, processing and presentation to activate and differentiate naïve T cells. Interestingly, we observed that nDCs showed highly activated phenotype, as supported by a significant augmentation in the expression of CD86 (p < 0.01) and CD40 (p < 0.01) than cDCs ( Fig. 2A,B). Further, abundant production of TNF-α (p < 0.05) by ELISA and considerable change in IFN-β was noted by RT-qPCR (Fig. 2C,D). In contrast, we observed significant decline in the levels of immunosuppressive cytokine TGF-β (p < 0.01) at mRNA level by RT-qPCR (Fig. 2E). Excitingly, nDCs exhibited significant (p < 0.01) enhancement in pinocytosis of the soluble antigen (HRP) (Fig. 2F) and phagocytosis of dextran-FITC and Mtb (p < 0.05) by confocal microscopy and CFUs, respectively (Fig. 2G,H). These results indicate that despite of highly activated phenotype, nDCs are efficient in antigen uptake. nDCs efficiently respond to innate stimuli. Next, we were curious to know if nDCs have not undergone tolerization and can still respond to other innate stimuli like TLRs. Noteworthy, nDCs showed significant improvement in the production of IL-6 and IL-12 than cDCs on signaling through different TLRs using their respective ligands; LPS for TLR-4 (IL-6: p < 0.001, IL-12: p < 0.001), Pam2Cys for TLR-2 (IL-6: p < 0.001, IL-12: p < 0.001), CpGODN for TLR-9 (IL-6: p < 0.01, IL-12: p < 0.05) and imiquimod for TLR-7 (IL-6: p < 0.05) (Fig. 3A,B). No change was observed in the case of poly I:C, a ligand for TLR-3. Further, we examined the effect of curdlan and trehalose-6, 6-dibehenate (TDB), a ligand for dectin-1 and mincle (C-type lectin receptors), respectively. Results showed the abundant release of IL-6 (p< 0.001) and IL-12 (p < 0.05) by nDCs than cDCs (Fig. 3C,D). Furthermore, triggering of nDCs through TLR-2 and TLR-4 showed upregulation of CD80 (p < 0.001) and CD40 (p < 0.001), as compared to control DCs (Fig. 3E,F). However, a marginal increase was noted with TLR-9 stimulation (Fig. 3G). These results signify the non-tolerogenic nature of nDCs.
Signaling of DCs through NOD-2 enhanced the release of NO. nDCs showed highly matured and activated phenotype. Therefore, we next investigated whether nDCs exhibit improved bactericidal activity than cDCs. Firstly, we measured NO since it is considered as a potent molecule responsible for bactericidal activity 20 . We noticed increase in the (p < 0.01) production of NO (Fig. 4A). Further, this was corroborated by detecting increased expression of iNOs (Fig. 4B). Similarly, higher expression of iNOs was also detected in the Mtb infected nDCs (Fig. 4C). Furthermore, these results were validated by greater production of NO (Fig. 4D) and expression of iNOs by LPS stimulated nDCs through colorimetric method, Western blotting and flow cytometry ( Fig. 4D-F). nDCs inhibit in vivo growth of intracellular pathogens. Since nDCs expressed activated phenotype, we were curious to know whether they also displayed better ability to restrict the intracellular proliferation of pathogens. We observed significantly lesser survival of Mtb in nDCs, as supported by reduced number of CFUs compared to cDCs (Fig. 5A). It is important to mention here that the bar graphs depict reduction in the growth of intracellular bacterium at 24h; normalized with the difference in Mtb uptake by nDCs after 4h of infection. Thus, proving that the change observed is due to restriction in the growth of Mtb and not owing to differential uptake.
The functionality of nDCs was ultimately examined in vivo in the experimental model of TB. The nDCs were adoptively transferred into mice, followed by infection with Mtb. As compared to cDCs, these animals showed significant (p < 0.01) decline in the Mtb burden in the spleen (Fig. 5B). Activation of T cells in Mtb model takes 14-15d for their effector functions. Therefore, to show the role of nDCs in reduction of the bacterial burden, Mtb challenged mice were sacrificed after 10d of infection. We were inquisitive to know whether the observed phenomenon was restricted to Mtb or to other pathogens, as well. Intriguingly, we noted significant decline in the CFUs of Salmonella typhimurium in the liver (p < 0.01) and spleen (p < 0.05) upon adoptive transfer of nDCs in mice (Fig. 5C,D); suggesting that the killing potency of nDCs was not restricted to Mtb alone. In addition, we further confirmed the involvement of NO in killing of Mtb. Mtb infected nDCs were cultured in the presence of iNOs inhibitor for 24 h. Interestingly, we observed the  21 . To evaluate their T cell priming capability, we carried out the adoptive transfer of nDCs loaded with OVA to OVA-sensitized mice. Interestingly, we observed that the inoculation of OVA loaded nDCs markedly increased the production of IFN-γ (p < 0.05), IL-2 (p < 0.05) and IL-17 (p < 0.05) ( Fig. 6A-C) than control DCs loaded with OVA. These results signify the optimum activation capacity of DCs. nDCs showed better potency to stimulate T cells. Next we assessed the ability of nDCs to vis-a-vis stimulate CD4 T cells and CD8 T cells. The T cells isolated from the spleen and lymph nodes of OVA sensitized mice were co-cultured with OVA pulsed-DCs. We observed that nDCs remarkably enhanced the activation of both CD4 T cells and CD8 T cells, as evidenced by substantially greater production of IFN-γ and IL-17 than cDCs ( Fig. 6D-G). Thus, illustrates that nDCs acquire enhanced potential to activate T cells. nDCs maturation and activation is mediated via IFN-αβ signaling. Ultimately, it was important for us to determine the NOD-2 driven mechanism responsible for the enhanced activity of nDCs. Cytokines like IL-6, IL-12, TNF-α and IFN-γ are known to regulate the differentiation and activation of DCs 10,22 . Noteworthy, NOD-2 recently has been shown to trigger the release of type-1 IFNs 23 . Further, type-1 IFNs have been shown to induce the maturation of DCs 24 . Taking into consideration of these facts, nDCs were differentiated in the presence of neutralizing/blocking Abs against IL-6, IL-12, IFN-γ , TNF-α and IFN-α β R. Remarkably, we observed that nDCs incubated with anti-IFN-α β R failed to acquire matured phenotype, as proven by significant (p < 0.01) downregulation in the expression of costimulatory molecule CD86 (Fig. 7A,B). In contrast, neutralization of IL-6, IL-12, IFN-γ and TNF-α showed no change (Fig. 7C-J). Likewise, blocking of IFN-α β R showed reduction (p > 0.001) in the NO production. This observation was corroborated by downregulation in the yield of iNOs by Western blotting (Fig. 7K,L).

Triggering of DCs through NOD-2 stimulates Stat-1 and Stat-4 and inhibits SOCS signaling pathway.
To gain further insight into the molecular mechanism underlying nDCs maturation and activation, we monitored the involvement of STAT signaling pathway, since it regulates the genes involved in the differentiation and maturation of DCs 25,26 . nDCs expressed higher phosphorylation of Stat-1 and Stat-4 than conventional DCs (Fig. 8A,B). Phosphorylation of Stat-1 induces the maturation of DCs 27,28 . Further, enhancement in the expression of Stat1 target gene IFN regulatory factor 1 (IRF1) was also noted (Fig. 8C). IRF-1 is known to induce the maturation and activation of DCs 29 . In addition, nDCs upon stimulation with LPS showed better phosphorylation of p38 molecules, which is also responsible for the activation of DCs (Fig. 8D) 30 . Furthermore, nDCs showed better nuclear translocation of NF-κ β (Fig. 8E). Suppressors of cytokine signaling (SOCS) molecules negatively regulate the expression of Stats to downregulate the activity of DCs 28 . Interestingly, nDCs showed reduction in the mRNA expression of SOCS-1 and SOCS-3 (Fig. 8F,G). These results collectively indicate that the higher expression of Stat-1 and Stat-4 along with downregulation of SOCS-1 and SOCS-3 is a mechanism responsible for improved function of nDCs.

Discussion
Bone marrow cells consist of macrophage and DC precursors, which give rise to circulating monocytes. During inflammation, monocytes migrate to the site of infection and differentiate into DCs and macrophages. Effect of different cytokines in skewing monocytes to DCs or macrophages is well established. Since bone marrow precursors express copious amount of PRRs, it is very likely that the interaction of these receptors with pathogenic moieties will influence their differentiation. Stimulation through NOD-2 efficaciously differentiates human monocytes to DCs in experimental model of leprosy and substantially improves their ability to cross-present antigen to CD8 T cells 15 .
In the current study, we have demonstrated a novel role of NOD-2 signaling in promoting the development of highly immunogenic nDCs. nDCs showed following major features: i) enhanced activation and maturation phenotype; ii) augmented bactericidal activity was through increased iNOs expression; iii) efficiently presented antigen to CD4 T cells and CD8 T cells; iv) improved capacity to inhibit in vivo growth of intracellular pathogens; v) the mechanism responsible for immunogenicity of nDCs was dependent on IFN-α β signaling pathways, leading to the activation of Stat-1 and Stat-4.
DCs functionality is determined by their immature and mature status 31 . Immature DCs are characterized by their ability of higher antigen uptake and lack the requisite signals for T cells activation. However, matured DCs lose their ability to uptake antigen. Further, they perform highly specialized function of priming T cells and therefore evoke adaptive immune response. nDCs exhibited augmented efficiency of antigen uptake and stimulation of T cells.
DCs differentiated in the presence of various TLR agonists are defective in their function 14 . Further, continuous exposure of DCs with antigen induces tolerization. Interestingly, unlike LPS (agonist of TLR-4), NGMDP (agonist of NOD-2) promoted the development of activated DCs 14 . Further, nDCs responded efficiently to different TLR agonists and upregulated the expression of costimulatory molecules and produced IL-6 and IL-12. Costimulatory molecules play a decisive role in deciding the anergy or activation of T cells 32,33 . IL-6 is a proinflammatory cytokine that is required at the time of infection and recruitment of cells to the inflammatory site. IL-12 is a hallmark cytokine to induce Th1 immunity 34 . DCs are inferior in bactericidal mechanism and restricting the growth of intracellular pathogens 2 . In contrast, nDCs chiefly activated Th1 cells and Th17 cells, exhibited enhanced bactericidal activity and were highly potent in killing Mtb. Further, they released more NO and displayed augmented levels of iNOs.
NOD-2 triggers IFN-α β production, which is a classical activator of STATs and phosphatidyl-inositol-3' kinase pathways 23,35 . Further, induction of interferon and STATs target genes are important components involved in DCs maturation and activation 27 . Noteworthy, nDCs showed maturation and activation involving phosphorylation of Stat-1, Stat-4 and p38 and induction in the expression of Stat-1 target gene IRF-1 and NF-κ B. Furthermore, blocking of IFN-α β resulted in the failure of nDCs to acquire highly activated phenotype, as evidenced by decrease in CD86 expression and NO release. STAT pathways are under the negative regulation of SOCS proteins. Importantly, NOD-2 triggered DCs showed decrease in mRNA expression of SOCS-1 and  For analysis of CD86, cells were gated on SSC + and CD11c + cells after the FSC and SSC gate was set. Bar graphs depict the integrated MFI (iMFI) for the CD86 expression. (K,L) nDCs cultured in the presence of blocking Abs to IFN-α β R showed decrease in the production of NO in the SNs and iNOs expression in whole cell lysate. Results depicted as mean ± SD are representative of 3 independent experiments. **p < 0.01, ***p < 0.001 ns: non-significant.
Scientific RepoRts | 6:27263 | DOI: 10.1038/srep27263 possible by obtaining large number of DCs through ex vivo generation of monocytes-driven DCs. Thus, it is imperative to identify factors that enhance the yield of DCs. It was extremely exciting for us to note that signaling through NOD-2 substantially expanded the pool of DCs with improved efficiency to stimulate innate immunity, as well as adaptive immunity by activating Th1 cells, Th17 cells and CD8 T cells. These cells play cardinal role in protecting against Mtb. Innate immunity is crucial in combating early infections, whereas adaptive immunity is necessary at later stage.
In essence, our study for the first time demonstrates a novel role of NOD-2 signaling in differentiating bone marrow precursors to highly immunogenic DCs. These DCs efficiently activated both innate as well as adaptive immunity that controlled the growth of Mtb. Finally, this strategy of stimulating DCs through NOD-2 may be extremely useful in bolstering immunity for treating infectious diseases.

In vitro infection with mycobacteria.
Mtb (H37Ra or H37Rv) were grown to mid log phase in 7H9 broth supplemented with glycerol, tween-80, albumin-10%, dextrose and catalase enriched medium. Liquid culture was grown for 12 d and aliquots were stored at − 80 °C till use. For infection, aliquots were rapidly thawed, washed twice with PBS (1X) and re-suspended in RPMI-FCS-10%. Bacteria were passed through a syringe and added to nDC cultures at a multiplicity of 1:5 infection and incubated for 4 h. Later, cells were washed extensively to remove extracellular bacteria.

CFU determination. The bacterial growth was quantified 24 h after infection. The cell supernatants (SNs)
were removed and saponin (0.1%) was added to lyse the cells and plating was done with 10 fold serial dilution on 7H11 agar plates and later kept at 37 °C in a humidified atmosphere. The colonies were enumerated after 3 wk. Antigen uptake. nDCs were harvested, washed, and then pulsed with HRP (50 μ g/ml). Pulse-chased experiments were conducted for 30-60 min and antigen uptake was arrested by adding chilled PBS (1X) followed by transferring cells on ice. The cells were washed extensively with ice cold PBS-FBS-1%. The cells were lysed using Tris-HCl (10 mM) and Triton X-100 (0.05%) for 30 min on ice, with intermittent vortexing. Intracellular HRP was estimated colorimetrically in the cell lysates using OPD-H 2 O 2 chromogen-substrate. The cells maintained at 4 °C were used as control. HRP activity in test samples was suitably normalized with controls.
For confocal analysis, nDCs were infected with GFP-Mtb (H37Ra) at (MOI 1:5) for 4 h. The cells were washed extensively (4X) with ice cold PBS (1X) and fixed with paraformaldehyde (4%). The cells were washed and placed on poly-L-lysine coated cover slips and imaged using Zeiss Confocal Laser Microscope. Results were examined by image analysis software.
For the estimation of bacterial uptake, nDCs were harvested and Mtb infection was given at MOI (1:5) for 4 h. It was followed by lysis of cells with 0.1% saponin and plating was done with 10 fold serial dilution on 7H11 agar plate. The colonies were enumerated by CFUs, 3 wk after incubation at 37 °C/5% CO 2 .

Real Time PCR.
To quantify RNA levels of nDCs, total RNA was isolated by Trizol reagent according to the manufacturer's instruction (Invitrogen, Carisbad, CA). RNA was quantified with the help of NanoDrop spectrophotometer. A260/A280 ratios of all samples were 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 using amplification grade DNase. Briefly, RNA samples (1 μ g) were incubated with DNase (1 U) for 15 min in the reaction buffer. After incubation, DNase activity was terminated by adding stop solution. Further, the samples were heated to 70 °C/10 min to inactivate DNase activity. Results were represented as relative expression (fold). Analysis was done by comparative Ct method. Real time PCR and data analysis were done by Realplex Mastercycler (Eppendorf, Hamburg, Germany). Western blotting. For iNOs, nDCs harvested on 6 d of culture were stimulated with LPS for 24 h or infected with Mtb for 4 h. Mtb infected DCs were washed to remove extracellular bacteria and cultured for 24 h. After 24 h, cells were 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 iNOs and actin (loading control). Blots were developed using chemiluminescence kit (Amersham Pharmacia Biotech, Buckinghamshire, UK). Blots were scanned using phosphoimager (Fujifilm, Tokyo, Japan) and images analyzed employing MultiGuage software. pStat-1, pStat-4 and p38 were detected in the lysate of nDCs harvested on 6 d of culture following the same protocol as mentioned above. The fold change in the phosphorylation of Stat-1, Stat-4 and p38 was calculated as a ration between phosphorylated and un-phosphorylated forms.
Adoptive transfer of nDCs to study bactericidal activity. nDCs were adoptively transferred into mice.
After 5 d, mice were challenged with Mtb (i.v) and Salmonella typhimurium (S. typhimurium) (i.p) and sacrificed on 10 d and 3 d, respectively. Later, Mtb burden was estimated in spleen and S. typhimurium in the liver and spleen.

In vivo polarization of T cells by nDCs.
The polarization of T cells was done as mentioned elsewhere 38 .
Mice were injected s.c. with OVA (100 μ g) in aluminum hydroxide (2 mg) dissolved in PBS (0.2 ml). Ten days later, nDCs incubated for 2 h with OVA (100 μ g/ml) were adoptively transferred s.c. into OVA-primed mice. On 7 d, the mice were sacrificed and lymph nodes were isolated and single cell suspension was prepared. These cells (2 × 10 5 /well) were then cultured with OVA (100 μ g/ml) for 24 h. Later, polarization of T cells was monitored by quantification of IFN-γ , IL-17 and IL-2 in the SNs by ELISA.

In vitro activation of CD4 T cells and CD8 T cells by nDCs.
Mice were sensitized by s.c. injection of OVA (100 μ g). After 7 d, lymph nodes were isolated and single cell suspension was prepared to purify CD4 T cells or CD8 T cells using magnetic associated cells sorting (MACS). Purified CD4 T cells or CD8 T cells were co-cultured with OVA loaded DCs at a ratio of 10:1 (T cells: DCs) for 5 d 38 . Later, IFN-γ and IL-17 were estimated in SNs by ELISA.
Statistics. Level of significance was performed by statistical analysis using unpaired student't test' by graph pad prism software.