Tri-mannose grafting of chitosan nanocarriers remodels the macrophage response to bacterial infection

Abstract

Background: Infectious diseases are still a leading cause of death and, with the emergence of drug resistance, pose a great threat to human health. New drugs and strategies are thus urgently needed to improve treatment efficacy and limit drug-associated side effects. Nanotechnology-based drug delivery systems are promising approaches, offering hope in the fight against drug resistant bacteria. However, how nanocarriers influence the response of innate immune cells to bacterial infection is mostly unknown.

Results: Here, we used Mycobacterium tuberculosis as a model of bacterial infection to examine the impact of mannose functionalization of chitosan nanocarriers (CS-NCs) on the human macrophage response. Both ungrafted and grafted CS-NCs were similarly internalized by macrophages, via an actin cytoskeleton-dependent process. Although tri-mannose ligands did not modify the capacity of CS-NCs to escape lysosomal degradation, they profoundly remodeled the response of M. tuberculosis-infected macrophages. mRNA sequencing showed nearly 900 genes to be differentially expressed due to tri-mannose grafting. Unexpectedly, the set of modulated genes was enriched for pathways involved in cell metabolism, particularly oxidative phosphorylation and sugar metabolism.

Conclusions: The ability to modulate cell metabolism by grafting ligands at the surface of nanoparticles may thus be a promising strategy to reprogram immune cells and improve the efficacy of encapsulated drugs.

Keywords: Chitosan nanocarriers; Host response; Macrophages; Mycobacterium tuberculosis; Surface grafting.

Fig. 1

Cellular uptake of chitosan NCs. a $\text{M}\mathrm{\varphi }\text{s}$ were incubated for 4 h with 10, 50, and 100 µg/ml Nile-Red-labelled CS-NCs. Particle internalization was assessed by FACS and the results expressed as the mean fluorescence intensity (MFI). b $\text{M}\mathrm{\varphi }\text{s}$ were cultured for 1, 4, or 18 h in the presence of 100 µg/ml fluorescent CS-NCs and particle internalization was quantitatively assessed by FACS. c $\text{M}\mathrm{\varphi }\text{s}$ were treated with 100 µg/ml fluorescent CS-NCs (red) for 1, 4, or 18 h and NC internalization visualized by confocal microscopy. DAPI (blue) was used to visualize nuclei. d 100 µg/ml Nile-Red-labelled CS-NC were incubated for 4 h with $\text{M}\mathrm{\varphi }\text{s}$, A549 epithelial cells, or HepG2 hepatocytes. NP uptake was quantitatively analyzed by FACS. e $\text{M}\mathrm{\varphi }\text{s}$ were incubated with 100 µg/ml fluorescent CS-NCs for 2 h with or without the pharmacological inhibitors nystatin, colchicine, cytochalasin D, or chlorpromazine. NC uptake was analyzed as previously described. Error bars represent the mean ± SD and significant differences between treatments are indicated by an asterisk, in which **p < 0.01 and ***p < 0.001

Fig. 2

Intracellular localization of chitosan NCs. a $\text{M}\mathrm{\varphi }\text{s}$ were cultured for 18 h in the presence of 100 µg/ml CS-NCs and intracellular visualization assessed by TEM. Yellow arrows: CS-NCs, Red arrows: NCs fusion. b $\text{M}\mathrm{\varphi }\text{s}$ were incubated with 100 µg/ml DiD-labelled CS-NCs (green) for 18 h, co-stained with LysoTracker (red) and DAPI (blue), and visualized by confocal microscopy. c Pearson correlation coefficient between CS-NCs and acidic compartments (Lysotracker positive). Each dot represents one single cell (n = 98). Error bars represent the mean ± SD. d $\text{M}\mathrm{\varphi }\text{s}$ were exposed to 100 µg/ml CS-NCs or latex beads for 18 h and incubated with Lysotracker Red. The intensity of the lysotracker staining was then quantified by FACS. Results are expressed as the mean fluorescence intensity (MFI). Significant differences between treatments and untreated controls are indicated by an asterisk, in which *p < 0.05. Error bars represent the mean ± SD

Fig. 3

Biological processes regulated by chitosan NCs on $\text{M}\mathrm{\varphi }\text{s}$. Transcriptome analysis was performed on $\text{M}\mathrm{\varphi }\text{s}$ isolated from three different donors and treated with 10 µg/ml CS-NCs for 18 h. a MA plot showing differentially expressed (DE) genes by CS-NCs relative to untreated controls. Genes with an FDR < 0.05 are shown in red. b Validation of the expression of selected candidate genes by ELISA. One representative experiment (out of three) is shown. Error bars represent the mean ± SD and significant differences between treatments are indicated by an asterisk, in which ***p < 0.001. c Significantly enriched biological processes (KEGG analysis) for genes regulated by CS-NC treatment relative to untreated controls. Interact. interaction, path. pathway

Fig. 4

Morphological characterization of tri-mannose-grafted chitosan NCs. a Schematic representation of a mannosylated CS-NC. b CryoTEM image of CS-NCs-tri. c Hydrodynamic diameter of CS-NCs-tri incubated at various concentrations of concanavalin A, assessed by dynamic light scattering

Fig. 5

Schematic Differentially-expressed genes upon chitosan NC and tri-mannose-grafted chitosan NC treatment. a $\text{M}\mathrm{\varphi }\text{s}$ were incubated with 100 µg/ml fluorescent CS-NCs and CS-NCs-tri. Internalization of the NCs was then assessed by FACS as in Fig. 1. b–e $\text{M}\mathrm{\varphi }\text{s}$ from three individual donors were treated for 18 h with 10 µg/ml CS-NCs or CS-NCs-tri. The differentially-expressed genes were then identified by mRNAseq. b Venn diagram showing the number of genes regulated by NC treatment relative to untreated controls. c Genes induced by CS-NCs or CS-NCs-tri that displayed > 1.5-fold difference (p < 0.05) in expression were plotted in a heat map. d Pathway classifications provided by GO of the differentially-expressed genes upon treatment with CS-NCs and CS-NCs-tri. e Pathway classifications provided by GO of the genes differentially-expressed only upon treatment with ungrafted CS-NCs. Sign. path. signaling pathway

Fig. 6

Grafting of tri-mmanose motifs on chitosan NCs modulates the response of M. tuberculosis-infected $\text{M}\mathrm{\varphi }\text{s}$. a $\text{M}\mathrm{\varphi }\text{s}$ were infected, or not, with M. tuberculosis and then treated with 100 µg/ml fluorescent CS-NC-tri for 18 h. Internalization of the CS-NC-tri was then assessed by FACS and the results expressed as the percentage uptake relative to uninfected cells treated with fluorescent CS-NC-tri (controls). The results are presented as the mean ± SD of three independent experiments. b Venn diagram showing differentially-expressed genes by M. tuberculosis-infected $\text{M}\mathrm{\varphi }\text{s}$ treated with CS-NCs or with CS-NCs-tri, as in Fig. 5b. c MA plot showing differentially expressed genes following CS-NC-tri treatment relative to untreated infected controls. Genes with an FDR < 0.05 are shown in red. d Graph of significantly enriched biological processes (KEGG analysis) for genes up-regulated (left graph) or down-regulated (right graph) by CS-NC-tri treatment in M. tuberculosis-infected $\text{M}\mathrm{\varphi }\text{s}$. The gray line indicates − log of p = 0.05. met. metabolism, phosph, phosphorylation, sign. path. signaling pathway