Research ArticleImmunology

Tolerogenic nanoparticles inhibit T cell–mediated autoimmunity through SOCS2

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Science Signaling  21 Jun 2016:
Vol. 9, Issue 433, pp. ra61
DOI: 10.1126/scisignal.aad0612
  • Fig. 1 Characterization of NPs.

    (A) Schematic representation of NPITE+Ins. (B) TEM analysis of the indicated NPs. (C) MIMO incorporation into NPs was quantified with a fluorescence-based peptide quantification kit as described in Materials and Methods. (D) Human embryonic kidney 293 cells transfected with a reporter plasmid encoding luciferase under the control of an AhR-responsive promoter were incubated with the indicated NPs, and luciferase activity was measured after 24 hours. Cotransfection with a thymidine kinase–Renilla construct was used for normalization purposes. Data are means ± SEM of one experiment representative of at least three independent experiments. Each experiment was performed with at least two biological samples. *P < 0.05 and **P < 0.01.

  • Fig. 2 NPITE+Ins administration suppresses T1D development in NOD mice.

    (A and B) Female NOD mice were left untreated (none) or were treated intraperitoneally weekly for 1 month with the indicated NPs. Treatment was started when the mice were 8 weeks old. Diabetes incidence (A) and glucose amounts (B) were measured at the indicated times. Data are means ± SEM of 12 to 15 mice per group from one experiment that is representative of three independent experiments. (C) Left: Histopathologic analysis of pancreas samples from the untreated and NP-treated mice described in (A) and (B). Analysis was performed when mice were 22 weeks old. Right: Analysis of the histological scores of mice treated with the indicated NP types. Data are means ± SEM of one experiment representative of at least three independent experiments. Each experiment was performed with at least eight biological samples. (D) Pancreatic lymph nodes from 22-week-old NOD mice treated with the indicated NP types were subjected to quantitative polymerase chain reaction (qPCR) analysis of the abundances of Rorc, Tbx21, and Foxp3 mRNAs, which were normalized to the abundance of Gapdh mRNA. (E to G) BDC2.5 NOD mice were treated weekly for 1 month with the indicated NPs. One week after the last NP administration, pancreatic lymph nodes were isolated and the percentages of IFN-γ+ and IL-17+ CD4+ T cells were analyzed by flow cytometry (E), the abundance of Foxp3 mRNA normalized with Gapdh mRNA was determined by qPCR analysis (F), and the percentage of FoxP3+ CD4+ T cells was determined by flow cytometric analysis (G). Numbers in the representative dot plots in (G) show the percentages of cells in the indicated gates. (H) NOD mice were treated once a week with the indicated NPs for 1 month. One week after the last NP administration, the percentages of DCs within pancreatic lymph nodes were analyzed by flow cytometry. cDC, classical dendritic cell. (I) DCs isolated from the NP-treated mice described in (H) were subjected to qPCR analysis of the abundance of Cyp1a1 mRNA normalized to that of Gapdh mRNA. (J) DCs isolated from the indicated NP-treated mice were treated with LPS for 24 hours and then were subjected to qPCR analysis of the abundances of Il6 and Il12a mRNAs normalized to that of Gapdh mRNA. Data are means ± SEM from one representative experiment of at least three independent experiments. All experiments were performed with at least three biological samples. *P < 0.05, **P < 0.01, and ***P < 0.001.

  • Fig. 3 NPITE+MIMO administration induces the generation of tolerogenic DCs.

    (A) TEM analysis of NPITE+MIMO uptake by splenic DCs after the indicated times in culture. Boxed areas in the images on the top row are shown under higher magnification in the bottom row. Images are from one representative experiment of two independent experiments, with each experiment performed with three biological samples. (B) Splenic DCs were incubated with the indicated NPs for 24 hours and then subjected to qPCR analysis of Cyp1a1 mRNA abundance normalized to that of Gapdh mRNA. (C) Splenic DCs incubated in vitro with the indicated NPs and activated with LPS for 24 hours were subjected to flow cytometric analysis of the relative abundances of the indicated cell surface markers. The red lines indicate the staining obtained from the control antibody, whereas the blue lines indicate specific antibody staining. Numbers inside the plots represent the percentages of cells within the indicated gates. (D and E) Splenic DCs were incubated in vitro with the indicated NPs and activated with LPS for 24 hours. The cells were then subjected to qPCR analysis of the abundances of Il6 and Il12a (D) and Il10 and Ido1 (E) mRNAs relative to that of Gapdh mRNA. (F to J) Splenic DCs were stimulated with LPS in the presence or absence of NPs for 24 hours and then cocultured with naïve BDC2.5+ CD4+ T cells for 3 days. T cell proliferation was evaluated by [3H]thymidine incorporation (F); IFN-γ and IL-17 production in culture medium was measured by enzyme-linked immunosorbent assay (ELISA) (G); Ifnγ and Il17 mRNA abundances were normalized to that of Gapdh mRNA (H); and the percentages of FoxP3, IFN-γ, and IL-17 expression in the gate of CD4+ T cells were analyzed by flow cytometry (I). Ratios of FoxP3+ to IFN-γ+ T cells (left) and of FoxP3+ to IL-17+ T cells (right) (J). Data in (B) to (H) are means ± SEM from one representative experiment of at least three independent experiments. Each experiment was performed with at least three biological samples. *P < 0.05, **P < 0.01, and ***P < 0.001.

  • Fig. 4 AhR activation by NPITE+Ins inhibits DC activation by inducing Socs2 expression.

    (A) Splenic DCs from NP-treated NOD mice and NP-treated BMDCs were subjected to gene expression analysis with NanoString nCounter arrays. Signaling pathways were targeted by NPITE+Ins in DCs. (B) Proposed effects of NPITE+Ins administration on NF-κB signaling in DCs. (C) Left: Splenic DCs were treated with the indicated NPs in the absence (−) or presence (+) of LPS, fractionated into cytoplasmic and nuclear fractions (Nu/Cyt), and then subjected to Western blotting analysis with antibodies specific for the indicated proteins. Western blots are from a single experiment and are representative of three independent experiments. Right: Densitometric analysis of the relative abundances of the indicated proteins. (D) Splenic DCs were treated with the indicated NPs and then subjected to qPCR analysis of Socs2 mRNA abundance relative to that of Gapdh mRNA. (E) Splenic DCs were treated with the indicated NPs in the absence or presence of LPS and then were analyzed by Western blotting with antibodies against the indicated proteins. Right: Densitometric analysis of the abundance of TRAF6 relative to that of GAPDH (glyceraldehyde-3-phosphate dehydrogenase) in the indicated cells. (F) Left: AhR-responsive elements (XRE-1, XRE-2, and XRE-3) in the mouse Socs2 promoter. Right: ChIP analysis of the interaction of AhR with XRE-1, XRE-2, and XRE-3 at the mouse Socs2 promoter in cells treated with the indicated NPs. (G) Splenic DCs from wild-type (WT) and AhR-d mice were treated with the indicated NPs and then were subjected to qPCR analysis of Socs2 mRNA abundance relative to that of Gapdh mRNA. (H) Left: Splenic DCs transfected with control siRNA or with Socs2-specific siRNA (siSocs2) were treated with the indicated NPs in the presence of LPS. The cells were fractionated and analyzed by Western blotting with a p65-specific antibody. Western blots are from one experiment and are representative of three experiments. Right: Densitometric analysis of the ratio of nuclear to cytoplasmic p65 abundance. (I) BMDCs transfected with control siRNA or with Socs2-specific siRNA were treated with the indicated NPs in the presence of LPS and then subjected to qPCR analysis of the abundances of Il6 and Il12a mRNAs normalized to that of Gapdh mRNA. (J) BMDCs transfected with control siRNA or with Socs2-specific siRNA were treated with NPITE+Ins and then cocultured with CD4+ T cells for 3 days. The T cells were then subjected to flow cytometric analysis of the percentages of IL-17+ and IFN-γ+ cells. Numbers in the plots represent the percentages of cells in the indicated gates. For all panels, a single experiment that is representative of three independent experiments is shown. Each experiment was performed with three biological samples. *P < 0.05, **P < 0.01, and ***P < 0.001.

  • Fig. 5 NPITE+GAD administration induces the generation of tolerogenic human DCs.

    (A to E) hDCs were incubated with NP, NPITE, NPGAD, or NPITE+GAD for 24 hours. (A) The cells were subjected to qPCR analysis of the abundance of CYP1A1 mRNA relative to that of GAPDH mRNA. (B and C) Gene expression was analyzed with NanoString nCounter arrays. Genes whose expression was increased in cells treated with NPITE+GAD compared to that in cells treated with NP are shown in blue, whereas genes whose expression was increased in cells treated with NPITE+GAD compared to that in cells treated with NP are shown in red. (D) hDCs treated with the indicated NPs were subjected to flow cytometric analysis of the relative abundances of the indicated cell surface markers. The red lines indicate the staining obtained from the control antibody, whereas the blue lines indicate specific antibody staining. Numbers inside the plots represent the percentages of cells within the indicated gates. (E) Cells of the human CD4+ T cell clone 325GAD were cocultured with hDCs prepared as in (D), and the amounts of IFN-γ in the cell culture medium were determined by ELISA. Data are from one representative experiment of at least three independent experiments, with each experiment performed with three biological samples. *P < 0.05 and ***P < 0.001.

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/9/433/ra61/DC1

    Fig. S1. NPITE+Ins administration suppresses CAD in NOD mice.

    Fig. S2. BMDCs loaded with NPITE+Ins suppress T1D in NOD mice.

    Fig. S3. NPs loaded with ITE activate AhR in BMDCs.

    Fig. S4. Effects of NPITE+Ins on Socs1 and Socs3 expression in DCs.

  • Supplementary Materials for:

    Tolerogenic nanoparticles inhibit T cell–mediated autoimmunity through SOCS2

    Ada Yeste, Maisa C. Takenaka, Ivan D. Mascanfroni, Meghan Nadeau, Jessica E. Kenison, Bonny Patel, Ann-Marcia Tukpah, Jenny Aurielle B. Babon, Megan DeNicola, Sally C. Kent, David Pozo, Francisco J. Quintana*

    *Corresponding author. Email: fquintana{at}rics.bwh.harvard.edu

    This PDF file includes:

    • Fig. S1. NPITE+Ins administration suppresses CAD in NOD mice.
    • Fig. S2. BMDCs loaded with NPITE+Ins suppress T1D in NOD mice.
    • Fig. S3. NPs loaded with ITE activate AhR in BMDCs.
    • Fig. S4. Effects of NPITE+Ins on Socs1 and Socs3 expression in DCs.

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    Citation: A. Yeste, M. C. Takenaka, I. D. Mascanfroni, M. Nadeau, J. E. Kenison, B. Patel, A.-M. Tukpah, J. A. B. Babon, M. DeNicola, S. C. Kent, D. Pozo, F. J. Quintana, Tolerogenic nanoparticles inhibit T cell–mediated autoimmunity through SOCS2. Sci. Signal. 9, ra61 (2016).

    © 2016 American Association for the Advancement of Science

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