Efficacy of SPG-ODN 1826 Nanovehicles in Inducing M1 Phenotype through TLR-9 Activation in Murine Alveolar J774A.1 Cells: Plausible Nano-Immunotherapy for Lung Carcinoma

Alveolar macrophages are the first line of defense against intruding pathogens and play a critical role in cancer immunology. The Toll-like receptor (TLR) family mediates an important role in recognizing and mounting an immune response against intruding microbes. TLR-9 is a member of the intracellular TLR family, which recognizes unmethylated CG motifs from the prokaryotic genome. Upon its activation, TLR-9 triggers downstream of the MyD-88-dependent transcriptional activation of NF-κB, and subsequently results in abundant inflammatory cytokines expression that induces a profound inflammatory milieu. The present exploratory investigation aimed at elucidating the potency of schizophyllan for entrapping ODN 1826 (SPG-ODN 1826)-mediated stimulation of TLR-9 in provoking an inflammatory-type response in murine alveolar macrophages. Schizophyllan (SPG), a representative of the β-glucan family, was used in the present study as a nanovehicle for endosomal trafficking of CpG ODN 1826. TEM analysis of SPG-ODN 1826 nanovehicles revealed that the prepared nanovehicles are spherical and have an average size of about 100 nm. Interestingly, SPG-ODN 1826 nanovehicles were competent in delivering their therapeutic payload within endosomes of murine alveolar macrophage (J774A.1) cells. Exposure of these nanovehicles within LPS stimulated J774A.1, resulted in a significant provocation of reactive oxygen species (ROS) (p < 0.01) in comparison to CpG ODN 1826 alone. Moreover, the formulated nanovehicles succeeded in generating a profound Th1-based cytokine profile constituted by enhanced expression of IFN-γ (p < 0.001) and IL-1β (p < 0.001) inflammatory cytokines. These findings clearly indicated the immunostimulatory potential of SPG-ODN 1826 nanovehicles for inducing the Th1-type phenotype, which would certainly assist in skewing M2 phenotype into the much-desired M1 type during lung cancer.


Introduction
Macrophages (Mφs), derived from the monocyte lineage, are a prerequisite for neutralizing the non-self constituents of the human body as well as the apoptotic or damaged cells The TEM micrograph of SPG-ODN 1826 nanovehicles formulated during our study showed near-spherical morphology and were found to be about 100 nm in diameter as evaluated through electron microscopy ( Figure 1A). EDX analysis was undertaken to ascertain the entrapment of CpG ODN 1826 with SPG. Figure 1B shows the elemental peaks of the SPG-ODN 1826 nanovehicle. The presence of a phosphorus peak, corresponding to the phosphorothioate (PS) backbone of the respective ODN, confirmed the efficient entrapment of CpG ODN 1826 within the SPG nanovehicle [27]. The EDX spectra show additional peaks corresponding to the different constituents of glass slides used during the analysis.
Schizophyllan or SPG is a constituent of the β-glucan family isolated from Schizophyllum commune and is recognized by Dectin-1 receptors on Mφ and other APCs [25]. SPG has been explored substantially for its applicability in delivering short sequences of nucleic acids intracellularly within immune cells, namely Mϕs and DCs. SPG-derived nanoparticles were documented to behave as a Dectin-1 non-agonist and deliver the immunostimulatory payload within endosomal TLR-9 [26]. Owing to these observations, we hypothesized that SPG nanovehicles entrapping CpG ODN 1826 could be used to instigate the desired M1 or inflammatory phenotype in murine alveolar Mϕs J774A.1 cells, and could thus represent plausible nano-immunotherapy for lung carcinoma.

Size, Morphology, and Entrapment of CpG ODN 1826 within SPG-ODN 1826 Nanovehicles
The TEM micrograph of SPG-ODN 1826 nanovehicles formulated during our study showed near-spherical morphology and were found to be about 100 nm in diameter as evaluated through electron microscopy ( Figure 1A). EDX analysis was undertaken to ascertain the entrapment of CpG ODN 1826 with SPG. Figure 1B shows the elemental peaks of the SPG-ODN 1826 nanovehicle. The presence of a phosphorus peak, corresponding to the phosphorothioate (PS) backbone of the respective ODN, confirmed the efficient entrapment of CpG ODN 1826 within the SPG nanovehicle [27]. The EDX spectra show additional peaks corresponding to the different constituents of glass slides used during the analysis.

SPG-ODN 1826 Nanovehicles Delivered TLR-9 Agonist within Endosomes
Owing to its intracellular localization and other associated intracellular factors, delivery of biological payloads to endosomes remains a challenging task. Confocal scanning laser microscopy (CSLM) analysis exhibited the relevance of SPGs in delivering TLR-9 agonist. The results illustrated that the plasma membrane of J774A.1 exhibited red fluorescence due to the affinity of FM4-64 dye toward the hydrophobic domains ( Figure 2A). The FAM-associated green fluorescence indicated the intracellular localization of SPG-ODN 1826 nanovehicles ( Figure 2B). The overlay image ( Figure 2C) of FM4-64 and FAM fluorescence exhibits a yellow color that indicates endosomal localization of SPGNPs within the alveolar macrophages.

SPG-ODN 1826 Nanovehicles Delivered TLR-9 Agonist within Endosomes
Owing to its intracellular localization and other associated intracellular factors, delivery of biological payloads to endosomes remains a challenging task. Confocal scanning laser microscopy (CSLM) analysis exhibited the relevance of SPGs in delivering TLR-9 agonist. The results illustrated that the plasma membrane of J774A.1 exhibited red fluorescence due to the affinity of FM4-64 dye toward the hydrophobic domains ( Figure 2A). The FAM-associated green fluorescence indicated the intracellular localization of SPG-ODN 1826 nanovehicles ( Figure 2B). The overlay image ( Figure 2C) of FM4-64 and FAM fluorescence exhibits a yellow color that indicates endosomal localization of SPGNPs within the alveolar macrophages.

Synthesized Nanovehicles Elucidated Non-Toxic Effects of J774A.1 Cells
The treatment of J744A.1 cells with SPG-ODN 1826 for 24 h was used to ascertain any plausible cytotoxicity on these cells. The results of the MTT assay clearly outlined that the formulated nanoformulation at different dilutions did not exert any potential cytotoxic effects on J774A.1 cells (Figure 3).

SPG-ODN 1826 Augmented ROS Levels within J774A.1 Cells
DCF-DA-mediated fluorescence was used to qualitatively and quantitatively assess the alterations within ROS levels induced by SPG-ODN 1826 in comparison to CpG ODN 1826. The photomicrographs indicated increased DCF-DA-mediated fluorescence in J774A.1 cells exposed to CpG ODN 1826, which was further increased in SPG-ODN 1826 nanovehicles-exposed cells ( Figure 4A). During the quantitative estimation of ROS, it was evident that with post-stimulation with LPS, the ROS level significantly increased within J774A.1 cells (positive control, p < 0.001) in comparison to the negative control. Treatment

Synthesized Nanovehicles Elucidated Non-Toxic Effects of J774A.1 Cells
The treatment of J744A.1 cells with SPG-ODN 1826 for 24 h was used to ascertain any plausible cytotoxicity on these cells. The results of the MTT assay clearly outlined that the formulated nanoformulation at different dilutions did not exert any potential cytotoxic effects on J774A.1 cells (Figure 3).

SPG-ODN 1826 Augmented ROS Levels within J774A.1 Cells
DCF-DA-mediated fluorescence was used to qualitatively and quantitatively assess the alterations within ROS levels induced by SPG-ODN 1826 in comparison to CpG ODN 1826. The photomicrographs indicated increased DCF-DA-mediated fluorescence in J774A.1 cells exposed to CpG ODN 1826, which was further increased in SPG-ODN 1826 nanovehicles-exposed cells ( Figure 4A). During the quantitative estimation of ROS, it was evident that with post-stimulation with LPS, the ROS level significantly increased within J774A.1 cells (positive control, p < 0.001) in comparison to the negative control. Treatment

SPG-ODN 1826 Augmented ROS Levels within J774A.1 Cells
DCF-DA-mediated fluorescence was used to qualitatively and quantitatively assess the alterations within ROS levels induced by SPG-ODN 1826 in comparison to CpG ODN 1826. The photomicrographs indicated increased DCF-DA-mediated fluorescence in J774A.1 cells exposed to CpG ODN 1826, which was further increased in SPG-ODN 1826 nanovehiclesexposed cells ( Figure 4A). During the quantitative estimation of ROS, it was evident that with post-stimulation with LPS, the ROS level significantly increased within J774A.1 cells (positive control, p < 0.001) in comparison to the negative control. Treatment with SPG-ODN 1826 nanovehicles significantly triggered the production of elevated levels of ROS (177 ± 4.35%; p < 0.01) compared to cells treated with CpG ODN 1826 (157 ± 1.52%) ( Figure 4B). with SPG-ODN 1826 nanovehicles significantly triggered the production of elevated levels of ROS (177 ± 4.35%; p < 0.01) compared to cells treated with CpG ODN 1826 (157 ± 1.52%) ( Figure 4B).

Discussion
Lung cancer is still the top cause of cancer-related mortality, and its treatment constitutes a major challenge. Regardless of the previous failure of immunotherapy in the treatment of lung cancer, increasing enthusiasm in cancer immunotherapy recently emerged [28]. The implication of nanotechnology has recorded a surge during the last couple of decades owing to the efficiency of nanovehicles in delivering the therapeutic cargo within different anatomical positions in the body [29][30][31]. In the present study, we aimed to explore the potency of schizophyllan, or SPG, as an established member of the β-glucan family and the mediated impelling of TLR-9 for prompting an inflammatorytype response in murine alveolar macrophages.

Discussion
Lung cancer is still the top cause of cancer-related mortality, and its treatment constitutes a major challenge. Regardless of the previous failure of immunotherapy in the treatment of lung cancer, increasing enthusiasm in cancer immunotherapy recently emerged [28].
The implication of nanotechnology has recorded a surge during the last couple of decades owing to the efficiency of nanovehicles in delivering the therapeutic cargo within different anatomical positions in the body [29][30][31]. In the present study, we aimed to explore the potency of schizophyllan, or SPG, as an established member of the β-glucan family and the mediated impelling of TLR-9 for prompting an inflammatory-type response in murine alveolar macrophages.
The β-glucan family comprises a group of β-D-glucose polysaccharides occurring in the bacterial or fungal cell walls [32]. Schizophyllan, or SPG, is an established member of the β-glucan family. Disintegrated β-glucan has been reported as a ligand for natural killer (NK)-like receptors (Dectin-1 receptors). Dectin-1 receptors are involved in the innate immune responses against fungi, but they can also recognize unidentified endogenous ligands on T cells that can consequently induce an efficient cellular response [33,34]. Interestingly, Dectin-1 acts as a major β-glucans receptor on a Mϕ and contributes to Mϕ-mediated cellular immune response against microbes and cancers [35].
TLR-9 is an endosomal Toll-like receptor, and its expression has been widely reported in various APCs including macrophages [36]. Using synergistic models, it was documented that agonist-mediated activation of TLR-9 either alone or in combinatorial approaches with chemotherapeutic and targeted therapeutic results in amelioration of tumor growth [9]. Furthermore, TLR9 agonists were shown to shift M2 Mφs to the M1 type, which, in accordance, can enhance an efficient immune response against cancer cells [9,16]. In addition to the finding that Dectin-1 regulates TLR-9-dependent gene expression, Dectin-1-mediated spleen tyrosine kinase (Syk) activation is required for TLR-9 trafficking to β-glucans [37]. In this context, we hypothesized that SPG β-glucan can activate TLR-9 and M1 Mφs to initiate an effective immune response against lung cancer cells.
Cancerous cells show augmented ROS levels, and treatment of such cells by prooxidants has emerged as an effective strategy [38,39]. Furthermore, an increase in intracellular ROS levels is also associated with deflated levels of growth factors, namely VEGF/VEGFR2, which not only results in apoptosis but also causes arrest in cell-cycle transition at the S phase [40]. In this study, the synthesized nanovehicles exhibited their potency in instigating the generation of ROS more significantly in comparison to the TLR-9 agonist, thereby indicating their relevance in stimulating the innate arm of the immune system. Importantly, J774A.1 cells pretreated with N-acetylcysteine (NAC) showed impaired ROS generation even after treatment with either TLR-9 agonist or SPG-ODN 1826 nanovehicles, reaffirming that ROS generation within alveolar Mφ was augmented by the treatment with TLR-9 agonist and nanovehicles.
Mφs within lungs, which are confined within the airspaces and are localized within the distal airway under homeostatic or stress conditions, play an indispensable role in inflammation and molding the type of immune response [41,42]. It was reported that alveolar macrophages play a complex and contradictory function in lung cancer. Proinflammatory cytokines secreted by alveolar macrophages were shown to improve anti-tumor functions. However, pro-tumor functions of alveolar macrophages in lung cancer were also indicated [43]. Generally, depending on their environment, Mφs can adopt numerous functional phenotypes; at least two phenotypes have been designated according to their role in inflammation. Pro-inflammatory M1 Mφs are induced by Th1 cytokines such as IFN-γ or IL-1β; alternatively, the M2 Mφs are activated by the Th2 cytokines [44]. In the tumor microenvironment, the M2-polarized Mφ constitutes the major population of tumor-associated macrophages (TAMs) penetrating the tumor. They promote proliferation and growth of cancer cells along with escalating metastasis and angiogenesis [45]. M2 Mφs are also competent in altering their phenotypes under the influence of inflammatory cytokines. In this study, the intracellular cytokine staining was conducted by incubating Mφs with various formulations at different time intervals to prevent their intracellular release from the cells. The intracellular cytokine staining results emphasized that SPG-ODN 1826 nanovehicles could efficiently stimulate TLR-9 as manifested by the profound increase in the positivity of IFN-γ and IL-1β in cells, which, however, was further augmented post-treatment with SPG-ODN 1826 nanovehicles. IFN-γ, a signature inflammatory cytokine, was repeatedly reported to induce the production of IFN-γ inducible protein (IP-10), an efficient anti-tumor molecule involved in attenuating tumor angiogenesis. Moreover, it is also well-known that IFN-γ acts as an important stimulus for skewing the Mφ phenotype into the M1 type, which exerts its anti-cancer efficacy through immune mediators, specifically iNOS, TNF, and IL-6 [43].
Collectively, the results of our study clearly demonstrated that TLR-9-agonist-mediated immunostimulatory potential was intensified by its entrapment within the schizophyllanbased nanovehicle. Furthermore, the nanovehicles succeeded in enhancing the bioavailability of TLR-9 agonist to its receptor within the endosomes of alveolar macrophages and induced substantial increases in ROS and Th1 inflammatory cytokines, which triggered the generation of an M1 base phenotype.

Materials
Mouse-specific CpG ODN 1826 with sequence 5 -T*C*C*A*T*G*A*C*G*T*T*C*C* T*G*A*C*G*T*T-3 (* represents a phosphorothioated backbone) was procured from Integrated DNA Technologies (San Diego, CA, USA). Schizophyllan, with a molecular weight of 450 kDa, was procured from Invivogen (San Diego, CA, USA).

Synthesis of SPG-ODN 1826 Nanovehicles
Complexation between SPG and CpG ODN 1826 was achieved without the use of poly-(A) tails in the ODN as per the protocol described earlier [27]. Initially, SPG was denatured into a single helix by being dissolved in DMSO at a concentration of 1 mg/mL and was then left for ultrasonic degradation for 4 h. The molar N/P ratio between SPG, CpG ODN, and PEI was fixed to 0.1886:10, respectively. After ultrasonic degradation, Tris-HCl and NaCl were added to the reaction mixture at final concentrations of 20 and 50 mM, respectively. CpG ODN was added to the reaction mixture and finally the pH was adjusted to neutral. The final composition of the reaction mixture per mL containing the SPG, PEI, and CpG ODN was 10.6 µM, 93.8 nM, and 2 µM, respectively. The reaction mixture was further incubated overnight at room temperature and then filtered using a 0.2 micron filter before making any measurements.

Energy Dispersive X-ray (EDX)
We carefully pipetted 50 µL of nanovehicle suspension on a clean borosilicate glass slide and were dried for 1 h under controlled temperature at 50 • C. The glass slide was then scanned using a JEOL 7610F SEM (Tokyo, Japan) at an operating voltage of 25 kV and the spectra were acquired with the aid of EDAX-TEAM software (Version 4.5) considering the sensitivity factor (k-factor) of the different elements.

TEM of SPG-ODN 1826 Nanovehicles
Synthesized nanovehicles were evaluated through TEM for determination of their size and morphology using standard procedures. Then, 20 µL of suspension was placed on the TEM grid and were left overnight. The dried grid was then visualized using a JEOL JEM2100 TEM (JEOL Ltd., Tokyo, Japan).

In Vitro Studies and Cell Culture Conditions
The alveolar murine macrophages (J774A.1) used in the study were obtained from the National Center for Cell Science, Pune, India. J774A.1 cells were maintained in DMEM-high glucose supplemented with 10% FBS and 1% antibiotic-antimycotic solution in a humidified atmosphere with 5% CO 2 at 37 • C. The cells were stimulated using LPS (1 µg/mL) for at least 4 h and served as the positive control. Flow cytometric analysis was conducted using a BD FACSCalibur flow cytometer (BD Biosciences, San Jose, CA, USA), where at least 10,000 events were analyzed for each experiment.

Evaluation of SPG-ODN 1826 Nanovehicles Induced Cell Cytotoxicity
The synthesized nanovehicles were evaluated for their cytotoxic potential on J774A.1 cells using the standard MTT assay as described previously [46]. During the assay, at least 10 4 J774A.1 cells were seeded in each well of a 96-well plate and were allowed to adhere in a standard culture environment overnight. LPS-stimulated cells were thereafter treated with either CpG ODN1826 or SPG-ODN 1826 nanovehicles (2 µm) and 96-well plate was further left undisturbed in ambient culture conditions for 24 h. Post-incubation media in each well were decanted and replaced with MTT (5 mg/mL, 100 µL) followed by another incubation of 4 h. Finally, DMSO was supplemented in each well to solubilize the formazan crystals before recording absorbance of SPG-ODN 1826 treated J774A.1 cells at 570 nm. Cell viability percentage was calculated using the following equation where A T is the absorbance of treated J774A.1 cells and A C is the absorbance of LPSstimulated, non-treated J774A.1 cells.

Determination of Intracellular Localization of SPG-ODN 1826 Nanovehicles
Intracellular localization of SPG-ODN 1826 was analyzed through confocal laser scanning microscopy (CLSM, Zeiss, LSM 780NLO, Oberkochen, Germany) as previously described [27]. In brief, 1.5 × 10 4 J774A.1 cells were transferred onto a single chamber cell culture slide (SP Lifesciences) and were allowed to adhere. The J774A.1 cells were stimulated using LPS as stated above. Post-stimulation, the cells were exposed to SPG-ODN 1826 nanovehicles for 6 h. Subsequently, the cells were stained using FM 4-64 following the manufacturer's instructions, and fixed using 4% paraformaldehyde. The slides were finally analyzed using a Carl Zeiss microscope (Zeiss, LSM 780NLO, Oberkochen, Germany).

Assessment of Intracellular ROS Levels
Qualitative assessment of the intracellular ROS levels with SPG-ODN 1826 treated J774A.1 cells was accomplished as described earlier [47]. For the assay, 1 × 10 4 J774A.1 cells/well were seeded in a 96-well plate and allowed to adhere under optimum culture conditions. Cells were stimulated with LPS and then treated with either CpG ODN1826 or SPG-ODN 1826 nanovehicles for 6 h. Thereafter, media in each well were decanted and replaced with 20 µM of DCFH-DA. The cells were further incubated for 30 min under standard culture conditions, after which they were washed gently and visualized in the FITC channel of a Floid imaging station (Thermo Scientific, Waltham, MA, USA). For quantitatively assessing the level of ROS, the above-stated procedure was used, and the DCF-DA-mediated fluorescence intensity was recorded at an excitation/emission ratio of 485/528 nm using a 96-well black bottom ELISA well through a Synergy H1 Hybrid fluorescent Reader (BioTek, Winooski, VT, USA).

Assessment of Inflammatory Cytokines Levels
The intracellular levels of signature inflammatory cytokines, namely IFN-γ and IL-1β, were estimated using cytometric bead array kits through the standard procedure. Initially, 1 × 10 6 J774A.1 cells/well were seeded in a 6-well plate and were allowed to adhere overnight. Post-stimulation with LPS, the cells were treated with SPG-ODN 1826 (dilution factor 10) nanovehicles and 2 µM of CpG ODN 1826 and incubated for a specified time (5 h for IFN-γ and 4 h for IL-1β). After incubation, the cells were treated with IFN-γ and IL-1β capture and detection beads following the manufacturer's protocol. Eventually, the cells were fixed using 4% paraformaldehyde and analyzed for PE-mediated fluorescence through flow cytometry using 575/40 nm excitation and 585/40 nm emission filters.

Statistical Analysis
The data illustrated are representative of three sets of independent experiments with their SEM and each experiment was conducted in triplets. The analysis of results was performed through one-way ANOVA followed by Tukey's multiple comparison post hoc test using GraphPad Prism (version 5.0). Differences of p < 0.05 between the treated groups were considered statistically significant.

Conclusions
The findings of the current study indicated that the use of the formulated SPG-ODN 1826 nanovehicles has the potential for further in vivo exploration owing to its immunostimulatory properties and because of its efficacy in inducing a prominent M1 or an inflammatory phenotype within the alveolar macrophages, which could pave the way for plausible immunotherapeutic interventions against lung carcinomas.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.

Conflicts of Interest:
The authors declare no conflict of interest.