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

Abstract

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.

Keywords: CpG ODN 1826; M1 and M2; Th1; inflammatory cytokines; lung cancer; schizophyllan.

Figure 1

Characterization of the synthesized nanovehicles by (A) transmission electron microscopy and (B) energy dispersive X-ray. The spectra elucidated the presence of a phosphorous peak, corresponding to the phosphorothioate (PS) backbone of the respective ODN. (Scale bar = 500 nm; magnification = 10,000×).

Figure 2

Confocal scanning laser photomicrograph exhibiting the endosomal internalization of SPG-ODN 1826 nanovehicles within J774A.1. (A) The plasma membrane is red due to the stain of FM 4-64 whereas (B) the internalized SPG-ODN 1826 nanovehicles appear green due to the presence of tagged FAM-CpG ODN 1826. (C) Endosomal localization of SPGNPs within alveolar macrophages J774A.1 cells was tracked using yellow fluorescence created by the overlay images of FM4-54 and FAM stain that resulted in a yellow color (indicated by arrows). Magnification = 32×, scale bar = 20 μm.

Figure 3

Cell viability of J774A.1 cells treated with different dilutions of synthesized SPG-ODN 1826 nanovehicles for 24 h using an MTT assay. The data presented are the mean ± SEM of three individual experiments, where each was performed in triplicate. Statistical significance between different groups was evaluated using one-way ANOVA where ns (non-significant) indicates p > 0.05.

Figure 4

Augmentation of ROS levels within J774A.1 cells treated with TLR-9 agonist and SPG-ODN 1826 nanovehicles. (A) Photomicrographs indicating augmented ROS in DCF-DA-positive J774A.1 cells treated with CpG ODN 1826 and nanovehicles. (B) Quantification of DCF-DA-mediated fluorescence within J774A.1 cells treated with TLR-9 agonist and formulated nanovehicles. Scale bar = 100 µm. Data presented are the mean ± SEM of three individual experiments, where each was performed individually thrice. Statistical significance between different groups was ascertained using one-way ANOVA and Tukey’s post hoc test where ** p < 0.01 and *** p < 0.001.

Figure 5

Intracellular staining of IFN-γ within cells. (A) PE-mediated positivity of J774A.1 against intracellular levels of IFN-γ as assessed during flow cytometric analysis; Black dots indicate IFN-γ negative cells, while red dots indicate IFN-γ positive cells. (B) quantified levels of IFN-γ within J774A.1 cells treated with CpG ODN-1826 and SPG-ODN 1826 nanovehicles with and without LPS stimulation. Data presented are mean ± SEM of three individual experiments, where each was performed individually thrice. Statistical significance between different groups was ascertained using one-way ANOVA and Tukey’s post hoc test where ** p < 0.01 and *** p < 0.001.

Figure 6

Intracellular staining of IL-1β within cells treated with various regimes formulated during the study. (A) PE-mediated positivity of J774A.1 against intracellular levels of IL-1β as assessed during flow cytometric analysis; Black dots indicate IL-1β negative cells, while red dots indicate IL-1β positive cells. (B) quantified levels of IFN-γ within J774A.1 cells treated with CpG ODN-1826 and SPG-ODN 1826 nanovehicles with and without LPS stimulation. Data presented are the mean ± SEM of three individual experiments, where each was performed individually thrice. Statistical significance between different groups was ascertained using one-way ANOVA and Tukey’s post hoc test where ** p < 0.01 and *** p < 0.001.