Specific immunosuppressive role of nanodrugs targeting calcineurin in innate myeloid cells

Abstract

Calcineurin (CN) inhibitors currently used to avoid transplant rejection block the activation of adaptive immune responses but also prevent the development of tolerance toward the graft, by directly inhibiting T cells. CN, through the transcription factors of the NFAT family, plays an important role also in the differentiation dendritic cells (DCs), the main cells responsible for the activation of T lymphocytes. Therefore, we hypothesized that the inhibition of CN only in DCs and not in T cells could be sufficient to prevent T cell responses, while allowing for the development of tolerance. Here, we show that inhibition of CN/NFAT pathway in innate myeloid cells, using a new nanoconjugate capable of selectively targeting phagocytes in vivo, protects against graft rejection and induces a longer graft acceptance compared to common CN inhibitors. We propose a new generation of nanoparticles-based selective immune suppressive agents for a better control of transplant acceptance.

Keywords: Drugs; Health sciences; Immune response; Immunology.

Graphical abstract

Figure 1

The activation of NFAT signaling pathway in DCs induce efficient T cell activation and graft rejection (A) Kaplan-Meier curve showing the percentage of WT recipients mice undergoing graft acceptance. WT females were transplanted with the skin of donor WT females or WT or NFATC2-deficient males; graft acceptance and rejection were monitored at the indicated time points until day 90 after transplantations. Long-rank (Mantel–Cox) statistical analysis was performed. ∗∗p < 0.001. (B) Kaplan-Meier curve showing the percentage of WT recipients undergoing graft acceptance. Recipient WT females were transplanted with DC-deficient or sufficient male skin or with female skin as control; graft acceptance and rejection were monitored at the indicated time points until day 80 after transplant. Long-rank (Mantel–Cox) statistical analysis was performed, ∗p < 0.05. (C) IFN-γ expression in skin grafts was measured 3 days after transplantation by qPCR. Fold changes (FC) in male over female skin grafts are shown both for WT and NFATc2-deficient skins. Values represent the mean +SE from 6 mice for each group. Statistical significance was determined with one-way analysis of variance, followed by Tukey’s multiple comparisons test, ∗p< 0.05, ns not statistically significant. (D) IFN-γ production by alloreactive T cells stimulated with WT or NFATc2-deficient DCs after 5 days of coculture. DCs were activated or not with LPS (1 μg/mL). Values represent the mean + SD of three independent experiments. Statistical significance was determined with one-way analysis of variance, followed by Tukey’s multiple comparisons test, ∗p< 0.05, ∗∗p< 0.001. (E) Efficiency of migration to draining lymph nodes of NFATC2-deficient and WT DCs measured by FITC painting. Values represent the absolute number of CD11c⁺MHCII⁺ FITC⁺ DCs from three independent experiments. Statistical significance was determined with student’s t test, ∗∗p< 0.001.

Figure 2

VIVIT nanoparticles inhibit the nuclear translocation of NFAT A20 cells expressing NFAT4-GFP fusion protein were pretreated with the indicated NPs (50 μg/mL) or with FK506 (10 ng/mL) and then stimulated with thapsigargin (TPG, 50nM) for 40 min. Nuclear translocation of NFAT4-GFP fusion protein was evaluated by confocal microscopy. Representative confocal images for each condition are shown (nuclei are marked in blue, scale bar = 25um) as well as the quantification of the NFAT4-GFP nuclear signal. Quantification data are presented as mean+SD. Statistical analysis was performed with one-way analysis of variance followed by Tukey’s multiple comparisons test, ∗∗∗∗p< 0.0001, ∗∗∗p< 0.001 n = 100 cells.

Figure 3

MYTS NPs are preferentially taken up by phagocytes in vivo The distribution of MYTS, HFn or PMDA NPs, 90 min after i.p. administration (100μg/mouse), was analyzed in the spleens of treated mice. The percentage of NP-positive DCs (CD11c⁺MHCII⁺ cells), macrophages (CD11b⁺ cells), neutrophils (Ly6G⁺CD11b⁺ cells), T (CD3⁺ cell) and B (CD19⁺ cells) lymphocytes are shown for each type of NP. Data are presented as mean +SE from three different mice.

Figure 4

MYTS NPs can escape the endosomal compartment (A) BMDCs were incubated for the indicated time points with 50 μg/mL of MYTS-FITC. Cells were then analyzed with flow cytometry for the internalization of NPs. DCs were identified as CD11c⁺MHCII⁺ cells. Data shown in the quantification graph are the mean +SD from three independent experiments. Statistical analysis was performed with one-way analysis of variance followed by Tukey’s multiple comparisons test, ∗∗p< 0.001. (B) TEM analysis of BMDCs treated with 50 μg/mL of MYTS for 90 min. Black arrows indicate NPs confined in endosomes; red arrows indicate NPs present in the cytoplasm. Panel insets represent higher magnification of the selected area. (C) Representative confocal microscopy images showing the intracellular distribution of MYTS-FITC in BMDCs after 5 h of co-culture; cell membrane is shown in red.

Figure 5

In vivo treatment with MYTS-VIVIT does not affect the capacity of T cells to respond to immunization (Upper panel) Scheme of the experiment. OT II mice were treated with MYTS-VIVIT or MYTS-PEG every other day or with FK506 every day for two weeks. After the two-week treatment, OVA-pulsed and LPS activated BMDCs were injected in the footpad of treated mice and draining lymph nodes collected after 72 h to assess activation of CD4⁺ OVA-specific T cell by means of IL-2 production. (Lower panel) IL-2 production by OVA-specific T cells from mice that received the indicated treatments. Data are shown as mean +SD and represent of two independent experiments 4 animals per group in total. Statistical analysis was performed with one-way analysis of variance followed by Tukey’s multiple comparisons test, ∗∗p< 0.001.

Figure 6

MYTS-VIVIT treatment induce long-term skin transplant acceptance (A) Upper panel, scheme of the experiment. Recipient mice were transplanted at day 0 and treated with NPs or FK506 every other day until day 50 after transplantation starting from day −1. Observations were performed at the indicated time points after surgery. Treatments were stopped at day 50 and mice observed again 20 days later (day 70). Lower panel, Kaplan-Meier curve showing the percentage of recipient mice undergoing graft acceptance. The arrow indicates the end of the treatment. Long-rank (Mantel–Cox) statistical analysis was performed, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001. (B) Upper panel, scheme of the experiment. Recipient mice were adoptively transferred at day 0 with OT II cells deprived of Treg cells and then transplanted with skin samples from K5mOVA mice. Transplanted recipients were treated with NPs or FK506 every other day starting from day −1 until day 50 after transplantation. Observation was performed at the indicated time points after surgery. Treatments were stopped at day 50 and mice observed again 20 days later (day 70). Lower panel, Kaplan-Meier curve showing the percentage of recipient mice undergoing K5-mOVA skin graft acceptance. The arrow indicates the end of the treatment. Long-rank (Mantel–Cox) statistical analysis was performed, ∗∗∗p < 0.001. (C) Upper panel, scheme of the experiment. Recipient mice were transplanted at day 0 with allogeneic skin and treated with NPs every other day or FK506 every day after transplantation starting from day −1. Inspections were performed daily starting from day 8 after transplantation. Lower panel, Kaplan-Meier curve showing the percentage of recipient mice undergoing graft acceptance. Long-rank (Mantel–Cox) statistical analysis was performed, ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001.