Research ArticleHEALTH AND MEDICINE

Modulating lung immune cells by pulmonary delivery of antigen-specific nanoparticles to treat autoimmune disease

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Science Advances  16 Oct 2020:
Vol. 6, no. 42, eabc9317
DOI: 10.1126/sciadv.abc9317
  • Fig. 1 Characterization of fabricated particles and the effect of intravenously delivered particles on EAE.

    (A) Representative SEM images of Nano-PLP, Micron-PLP, and Nano-OVA particles. (B) Size, charge, and PLP139–151 loading of particles (n = 3 to 4 batches; means ± SD). Representative in vivo imaging system (IVIS) images of (C) lung and (D) liver of mice at day 14 p.i. after intravenous delivery of 1.5 mg of Cy5.5+ Nano-PLP and Cy5.5+ Micron-PLP particles at day 7 p.i. Cy5.5 Nano-OVA particles as control. (E) Particle-positive cells in the liver, lung, and spleen at day 14 p.i. [two-way analysis of variance (ANOVA) followed by Bonferroni; n = 5 mice per group]. (F) EAE clinical scores and (G) cumulative EAE clinical scores for intravenously administered particles (two-way ANOVA followed by Tukey’s multiple comparison; n = 12 mice for Nano-PLP; n = 11 mice for Micron-PLP and Nano-OVA). (F) ##P < 0.01 Nano-PLP and Micron-PLP versus Nano-OVA, and *P < 0.05 Nano-PLP versus Micron-PLP. Numbers of (H) myeloid cells, (I) CD4+ T cells, and (J) B cells in the CNS of EAE mice at day 14 p.i. after particle delivery at day 7 p.i. (one-way ANOVA followed by Tukey’s multiple comparison; n = 3 mice per group). *P < 0.05, **P < 0.01, and ***P < 0.001.

  • Fig. 2 Modulation of EAE mice by intratracheally delivered Nano-PLP particles.

    (A) Timeline of immunization, intratracheal particle administration, IVIS, and FACS analysis. IVIS images of (B) lung and (C) liver following Cy5.5+ particle administration (1.5 mg per mouse; PBS control). (D) EAE clinical scores of particles administered intratracheally (Nano-PLP-IT and Nano-OVA-IT) or intravenously (Nano-PLP-IV) (two-way ANOVA followed by Tukey’s multiple comparison for Nano-PLP-IT versus other groups; n = 12 mice for Nano-PLP-IV; n = 11 mice for Nano-PLP-IT; n = 10 mice for Nano-OVA-IT). (E) EAE clinical scores of intratracheally delivered Nano-PLP (0.5, 1.0, or 1.5 mg per mouse) or phosphate-buffered saline (PBS) (two-way ANOVA followed by Tukey’s multiple comparison for significant difference of 1.5 mg per mouse versus other groups; n = 11 mice for Nano-PLP; n = 8 mice for PBS). (F to H) FACS analysis of immune cells in the CNS of EAE mice at day 14 p.i. (F) Numbers of macrophages, DCs, neutrophils, and monocytes. Numbers of (G) macrophages and (H) DCs expressing CD86 and major histocompatibility complex class II (MHCII) (unpaired two-tailed t test; n = 5 mice per group). (I) Weekly intratracheal administration of 1.0 mg per mouse (multiple t test followed by Holm-Sidak’s multiple comparison; n = 13 for Nano-PLP; n = 9 for Nano-OVA). *P < 0.05, **P < 0.01, and ***P < 0.001.

  • Fig. 3 Intratracheally delivered Nano-PLP particles influence population and phenotypes of lung immune cells.

    Lung immune cells were assessed following intratracheally delivered Nano-PLP and Nano-OVA particles for their association with particles and their phenotypes at day 14 p.i. after delivery of 1.5 mg of particles at day 7 p.i. (A) Representative tSNE dimension 1 and 2 plots of the lung myeloid compartment generated from concatenated CD45+ cells derived from flow cytometric data overlaid manually on the gated cell populations. (B) Number of lung immune cells, including AMs, CD11b+ IM, CD11c+ IMs, cDCs, Mo DCs, Inf. Macs, Inf. Monos, and neutrophils. (C) Representative heatmap of tSNE shows the association of immune cells with Nano-PLP or Nano-OVA particles. (D) Number of lung immune cells associated with particles, including AMs, CD11b+ IM, CD11c+ IMs, cDCs, Mo DCs, Inf. Macs, Inf. Monos, and neutrophils. (E) Representative heatmap of tSNE shows MHCII expression on immune cells. (F) Number of MHCII+CD86 expression on the lung immune cells, including AMs, CD11b+ IM, CD11c+ IMs, cDCs, Mo DCs, Inf. Macs, Inf. Monos, and neutrophils. (B, D, and F) Unpaired two-tailed t test (n = 5 mice per group). *P < 0.05, **P < 0.01, and ***P < 0.001.

  • Fig. 4 Intratracheally delivered Nano-PLP particles influence the population and phenotypes of APCs in the Med LNs.

    FACS analysis of APCs in the Med LNs of EAE mice at day 14 p.i. after intratracheal delivery of 1.5 mg of particles at day 7 p.i. (A) Number of macrophages and DCs in the Med LNs. (B) Population of CD86+ macrophage and DCs in the Med LNs. (C) Population of particle-associated macrophages and DCs in the Med LNs. (D) CD86 expression of macrophages and DCs with or without Nano-PLP particles (Nano-PLP+, Nano-PLP) in the Med LNs. (A to D) Unpaired two-tailed t test was performed (n = 5 mice per group). *P < 0.05 and ***P < 0.001.

  • Fig. 5 Intratracheally delivered Nano-PLP particles influence the population of CD4+ T and B cells, expression of IFN-γ, IL-17, Foxp3, and gene expressions in the CNS, lung, and Med LNs.

    The CNS, lung, and Med LNs were harvested from EAE mice at day 14 p.i. after intratracheal delivery of 1.5 mg of particles at day 7 p.i. Numbers of (A) CD4+ T cells and (B) B cells. Numbers of (C) IFN-γ, (D) IL-17, and (E) Foxp3 expressing CD4+ T cells. (A to E) Unpaired two-tailed t test (n = 5 mice per group). (F to I) Real-time polymerase chain reaction (PCR) quantification of relative expression of chemokine [(F) CCL5, (G) CXCL1, (H) CCL21B, and (I) CCL19] genes. Expression of each gene was calculated relative to the expression of housekeeping gene, GAPDH. Unpaired t test (n = 5 mice per group). *P < 0.05 and ***P < 0.001.

  • Fig. 6 Reduction in CD4+ T cell proliferation by lung MHCII+ cells associated with intratracheally delivered Nano-PLP particles.

    (A) Schematic of experiment showing Nano-PLP particles delivered intratracheally at day 7 p.i. (no particle control), with isolation and sorting of lung MHCII+ cells with or without Nano-PLP particles (Nano-PLP+/−) by FACS at day 13 p.i. These isolated Nano-PLP+/− MHCII+ cells were cultured with CD4+ T cells from non-lung draining lymph nodes and spleen of EAE mice that had not been treated with Nano-PLP particles. (B) CD4+ T cell proliferation data including (a) T cell only (from EAE mice without particle treatments), (b) MHCII+ cells with Nano-PLP particles (Nano-PLP+), (c) MHCII+ cells without Nano-PLP particles (Nano-PLP), and (d) MHCII+ cells (from EAE mice without particle treatments).

Supplementary Materials

  • Supplementary Materials

    Modulating lung immune cells by pulmonary delivery of antigen-specific nanoparticles to treat autoimmune disease

    Eiji Saito, Stephen J. Gurczynski, Kevin R. Kramer, Carol A. Wilke, Stephen D. Miller, Bethany B. Moore, Lonnie D. Shea

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