Curcumin Nanoparticles Enhance Mycobacterium bovis BCG Vaccine Efficacy by Modulating Host Immune Responses

Nanocurcumin enhances BCG vaccine efficacy in H37Rv-challenged mice. Mice were distributed into 4 groups: (i) naive C57BL/6 mice, (ii) nanocurcumin (NC)-treated mice, (iii) BCG-immunized and PBS-treated mice, and (iv) BCG-immunized and nanocurcumin-treated mice. (a) Schematic representation of the experiment. Mice were immunized with BCG (subcutaneously) and then injected with nanocurcumin for 30 days, followed by a resting period of another 30 days. Mice were then challenged with H37Rv via the aerosol route, with a low-dose inoculum of approximately 110 CFU per mouse. After that, mice were euthanized at various time points (15, 30, 75, and 110 days), and lungs and spleen were harvested and assessed for bacterial burden. (b) CFU counts in lungs at different time intervals. (c) CFU counts in spleen at different time intervals. (d, left) Photomicrographs of lung histological sections (6 μm) of different experimental groups after 75 days of infection, stained with hematoxylin and eosin. Arrows denote the granulomatous region. (Right) Graphical representation of the percentages of granulomatous regions in different experimental groups. All data are representative of results from 3 independent experiments with 5 mice from each experimental group at each time point. All values are represented as means ± SD. Statistical analyses were done by ANOVA with Tukey’s post hoc analysis. In panels b and c, comparisons were done between the group receiving BCG, nanocurcumin, and H37Rv and all other experimental groups. * denotes a P value of ≤0.05. Experimental groups are (i) H37Rv, (ii) nanocurcumin plus H37Rv, (iii) BCG plus H37Rv, and (iv) BCG plus nanocurcumin and H37Rv.

Nanocurcumin activates APCs in lungs of BCG-vaccinated mice. Mice were treated as described in the legend to Fig. 2a. At 75 days postinfection, lungs were harvested, and single-cell suspensions were made and cultured overnight, followed by staining and flow cytometry. Primarily monocytes were gated based on forward scatter versus side scatter (SSC), and monocytes and other cell types were identified by specific antibodies. (a) Pseudocolor plot and bar diagrams of CD11b⁺ and CD11c⁺ APCs in lungs. (b) Activation status of APCs. Pseudocolor plots and bar diagrams of CD11b⁺ CD86⁺ and CD11c⁺ CD86⁺ APCs in lungs are shown. (c) Bar diagrams of IL-12-, IL-10-, IL-6-, and iNOS-producing APCs. All data are representative of results from 3 independent experiments with 5 mice from each experimental group at each time point. All values are represented as means ± SD. Statistical analyses were done by ANOVA with Tukey’s post hoc analysis. * denotes a P value of ≤0.05. Experimental groups are (i) uninfected, (ii) H37Rv, (iii) nanocurcumin (NC) plus H37Rv, (iv) BCG plus H37Rv, and (v) BCG plus nanocurcumin and H37Rv.

Nanocurcumin treatment increases the proliferation and activation of antigen-specific CD4⁺ and CD8⁺ T cells in BCG-immunized animals. Mice were treated as described in the legend to Fig. 2a. At 72 days postinfection, mice were injected with BrdU. Seventy-two hours after injection of BrdU, spleens were harvested, and single-cell suspensions were made. Cells were cultured overnight with M. tuberculosis lysate (CSA) stimulation to assess antigen-specific immune responses. These cells were stained with anti-CD3, -CD4, -CD8, -CD69, and -BrdU antibodies, followed by flow cytometry. For FACS analysis, we first isolated the CD3 and then the CD4 or CD8 populations, and within those populations, we then gated BrdU⁺ and CD69⁺ populations. (a) CD4 T cell proliferative status, as shown by pseudocolor and bar diagrams of CD4⁺ BrdU⁺ T cells. (b) CD8 T cell proliferative status, as shown by pseudocolor plots and bar diagrams of CD8⁺ BrdU⁺ T cells. (c) CD4 T cell activation status, as shown by pseudocolor plots and bar diagrams of CD4⁺ CD69⁺ T cells. (d) CD8 T cell activation status, as shown by pseudocolor plots and bar diagrams of CD8⁺ CD69⁺ T cells. All data are representative of results from 3 independent experiments with 5 mice from each experimental group at each time point. All values are represented as means ± SD. Statistical analyses were done by ANOVA with Tukey’s post hoc analysis. * denotes a P value of ≤0.05. Experimental groups are (i) H37Rv, (ii) nanocurcumin (NC) plus H37Rv, (iii) BCG plus H37Rv, and (iv) BCG plus nanocurcumin and H37Rv.

Nanocurcumin enhances BCG vaccine efficacy by increasing the T_CM/T_EM cell ratio in spleen. Mice were treated as described in the legend to Fig. 2a. At 75 days postinfection, spleens were harvested, and single-cell suspensions were made. Cells were cultured overnight with the M. tuberculosis lysate (CSA) to assess antigen-specific immune responses. These cells were stained with anti-CD3, -CD4, -CD8, -CD44, -CD62L, and -CCR7 antibodies, followed by flow cytometry. Different memory subsets, including naive T cells (CD4⁺ CD44⁻ CD62L⁻/CD4⁺ CD44⁻ CCR7⁻ or CD8⁺ CD44⁻ CD62L⁻/CD8⁺ CD44⁻ CCR7⁻), T_CM cells (CD4⁺ CD44⁺ CD62L⁺/CD4⁺ CD44⁺ CCR7⁺ or CD8⁺ CD44⁺ CD62L⁺/CD8⁺ CD44⁺ CCR7⁺), and T_EM cells (CD4⁺ CD44⁺ CD62L⁻/CD4⁺ CD44⁺ CCR7⁻ or CD8⁺ CD44⁺ CD62L⁻/CD8⁺ CD44⁺ CCR7⁻), were analyzed by flow cytometry. (a) Contour plots and bar diagrams of different (CD62L-gated) CD4⁺ T memory subsets in spleens of different experimental groups. (b) Contour plots and bar diagrams of different (CD62L-gated) CD8⁺ T memory subsets in spleens of different experimental groups. (c) Contour plots and bar diagrams of different (CCR7-gated) CD4⁺ T memory subsets in spleens of different experimental groups. (d) Contour plots and bar diagrams of different (CCR7-gated) CD8⁺ T memory subsets in spleens of different experimental groups. All data are representative of results from 3 independent experiments with 5 mice from each experimental group at each time point. All values are represented as means ± SD. Statistical analyses were done by ANOVA with Tukey’s post hoc analysis. * denotes a P value of ≤0.05. Experimental groups are (i) H37Rv, (ii) nanocurcumin (NC) plus H37Rv, (iii) BCG plus H37Rv, and (iv) BCG plus nanocurcumin and H37Rv.

Nanocurcumin enhances the T_CM/T_EM cell ratio in lungs of BCG-immunized mice. Mice were treated as described in the legend to Fig. 2a. At 75 days postinfection, lungs were harvested, and single-cell suspensions were made. Cells were cultured overnight with the M. tuberculosis lysate (CSA) and stained to assess antigen-specific immune responses. Cells were stained with anti-CD3, -CD4, -CD8, -CD44, -CD62L, and -CCR7 antibodies, followed by flow cytometry. Different memory subsets, including naive T cells (CD4⁺ CD44⁻ CD62L⁻/CD4⁺ CD44⁻ CCR7⁻ or CD8⁺ CD44⁻ CD62L⁻/CD8⁺ CD44⁻ CCR7⁻), T_CM cells (CD4⁺ CD44⁺ CD62L⁺/CD4⁺ CD44⁺ CCR7⁺ or CD8⁺ CD44⁺ CD62L⁺/CD8⁺ CD44⁺ CCR7⁺), and T_EM cells (CD4⁺ CD44⁺ CD62L⁻/CD4⁺ CD44⁺ CCR7⁻ or CD8⁺ CD44⁺ CD62L⁻/CD8⁺ CD44⁺ CCR7⁻), were analyzed by flow cytometry. (a) Contour plots and bar diagrams of different (CD62L-gated) CD4⁺ T memory subsets in lungs of different experimental groups. (b) Contour plots and bar diagrams of different (CD62L-gated) CD8⁺ T memory subsets in lungs of different experimental groups. (c) Contour plots and bar diagrams of different (CCR7-gated) CD4⁺ T memory subsets in spleens of different experimental groups. (d) Contour plots and bar diagrams of different (CCR7-gated) CD8⁺ T memory subsets in spleens of different experimental groups. All data are representative of results from 3 independent experiments with 5 mice from each experimental group at each time point. All values are represented as means ± SD. Statistical analyses were done by ANOVA with Tukey’s post hoc analysis. * denotes a P value of ≤0.05. Experimental groups are (i) H37Rv, (ii) nanocurcumin (NC) plus H37Rv, (iii) BCG plus H37Rv, and (iv) BCG plus nanocurcumin and H37Rv.