Synthetic Nanoparticles That Promote Tumor Necrosis Factor Receptor 2 Expressing Regulatory T Cells in the Lung and Resistance to Allergic Airways Inflammation

Abstract

Synthetic glycine coated 50 nm polystyrene nanoparticles (NP) (PS50G), unlike ambient NP, do not promote pulmonary inflammation, but instead, render lungs resistant to the development of allergic airway inflammation. In this study, we show that PS50G modulate the frequency and phenotype of regulatory T cells (Treg) in the lung, specifically increasing the proportion of tumor necrosis factor 2 (TNFR2) expressing Treg. Mice pre-exposed to PS50G, which were sensitized and then challenged with an allergen a month later, preferentially expanded TNFR2⁺Foxp3⁺ Treg, which further expressed enhanced levels of latency associated peptide and cytotoxic T-lymphocyte associated molecule-4. Moreover, PS50G-induced CD103⁺ dendritic cell activation in the lung was associated with the proliferative expansion of TNFR2⁺Foxp3⁺ Treg. These findings provide the first evidence that engineered NP can promote the selective expansion of maximally suppressing TNFR2⁺Foxp3⁺ Treg and further suggest a novel mechanism by which NP may promote healthy lung homeostasis.

Keywords: PS50G; animal model; asthma; lung; lymph nodes; nanoparticles; tumor necrosis factor 2.

Figure 1

PS50G instillation selectively increases lung CD3⁺CD4⁺CD25⁺ cells that are TNFR2⁺Foxp3⁺. Naïve mice (n = 5–7 per group per time point) received PS50G intratracheally on day 0 or saline as control. Samples were collected on days 1, 3, 7, and 30. (A) Stained lung cells were gated on viable CD3⁺CD4⁺CD25⁺ and CD3⁺CD4⁺CD25⁻ cells, followed by gating on TNFR2 co-expressed with Foxp3. Representative FACS contour plots showing TNFR2⁺ cells in the lung on day 3 from PS50G treated mice. Percentages of (B) CD3⁺CD4⁺CD25⁺ cells; (C) TNFR2⁺Foxp3⁺ regulatory T cells (Treg); (D) TNFR2^–Foxp3⁺ Treg; and (E) TNFR2⁺Foxp3⁻ Teff. Data represent the mean ± SEM of at least three experiments. ***p < 0.001.

Figure 2

PS50G instillation selectively increases lung-draining lymph nodes (LN) CD3⁺CD4⁺CD25⁺ cells that are TNFR2⁺Foxp3⁺ on day 7. Naïve mice (n = 5–7 per group per time point) received PS50G intratracheally on day 0 or saline as control. Samples were collected on days 1, 3, 7, and 30. (A) Stained lung-draining LN cells were gated on viable CD3⁺CD4⁺CD25⁺ and CD3⁺CD4⁺CD25⁻ cells, followed by gating on TNFR2 co-expressed with Foxp3. Representative FACS contour plots showing TNFR2⁺ cells in the lung-draining LN at day 3 from PS50G-treated mice. Percentages of (B) CD3⁺CD4⁺CD25⁺ cells; (C) TNFR2⁺Foxp3⁺ regulatory T cells (Treg); (D) TNFR2^–Foxp3⁺ Treg; and (E) TNFR2⁺Foxp3⁻ Teff. Data represent the mean ± SEM of at least three experiments. ***p < 0.001.

Figure 3

PS50G instillation selectively increases lung CD3⁺CD4⁺CD25⁺ T cells that are TNFR2⁺Foxp3⁺ in allergic airway inflammation mouse model. (A) Stained lung cells were gated on viable CD3⁺CD4⁺CD25⁺ and CD3⁺CD4⁺CD25⁻ cells, followed by gating on tumor necrosis factor 2 co-expressed with Foxp3. Percentages of (B) TNFR2⁺Foxp3⁺ regulatory T cells (Treg); (C) TNFR2^–Foxp3⁺ Treg and (D) TNFR2⁺Foxp3⁻ Teff. Ratios of (E,G) TNFR2⁺Foxp3⁺ Treg and (F,H) TNFR2^–Foxp3⁺ Treg to Foxp3⁻ Teff. Data represent the mean ± SEM of at least three experiments with four to six mice per group. *p < 0.05 and ***p < 0.001 compared with saline negative control group (Sal/Sal/Sal); ^#p < 0.01, compared with OVA positive control group (Sal/OVA/OVA).

Figure 4

PS50G instillation selectively increases lung-draining lymph nodes CD3⁺CD4⁺CD25⁺ that are TNFR2⁺Foxp3⁺ in allergic airway inflammation mouse model. (A) Stained lung cells were gated on viable CD3⁺CD4⁺CD25⁺ and CD3⁺CD4⁺CD25⁻ cells, followed by gating on tumor necrosis factor 2 co-expressed with Foxp3. Percentages of (B) TNFR2⁺Foxp3⁺ regulatory T cells (Treg); (C) TNFR2⁻Foxp3⁺ Treg; and (D) TNFR2⁺Foxp3⁻ Teff. Ratios of (E,G) TNFR2⁺Foxp3⁺ Treg and (F,H) TNFR2⁻Foxp3⁺ Treg to Foxp3⁻ Teff. Data represent the mean ± SEM of at least three experiments with four to six mice per group. *p < 0.05, **p < 0.01, ***p < 0.001 compared with saline negative control group (Sal/Sal/Sal); ^#p < 0.05, compared with OVA positive control group (Sal/OVA/OVA).

Figure 5

PS50G instillation increases the percentages of Ki67⁺ cells within lung TNFR2⁺Foxp3⁺ regulatory T cells (Treg) during allergic airway inflammation. Stained lung and lung-draining lymph nodes (LN) cells were gated as in Figures 3A and 4A, respectively followed by gating on Ki67. Percentages of (A) lung Ki67⁺ and (B) lung-draining LN Ki67⁺ within TNFR2⁺Foxp3⁺ Treg; TNFR2⁻Foxp3⁺ Treg and TNFR2⁺Foxp3⁻ Teff. Data represent the mean ± SEM of at least three experiments with four to six mice per group. ^#p < 0.05, compared with OVA positive control group (Sal/OVA/OVA). The dashed lines denote the percentages of cells expressing the respective markers derived from the saline negative control group (Sal/Sal/Sal).

Figure 6

PS50G instillation increases the percentages of lung LAP⁺ and lung-draining lymph nodes (LN) CTLA-4⁺ cells within TNFR2⁺Foxp3⁺ regulatory T cells (Treg) during allergic airway inflammation. Stained lung cells and lung-draining LN cells were gated as in Figures 3A and 4A, respectively, followed by gating on latency associated peptide and CTLA-4. Percentages of (A) lung LAP⁺; (B) lung-draining LN LAP⁺; (C) lung CTLA-4⁺; and (D) lung-draining LN CTLA-4⁺ within TNFR2⁺Foxp3⁺ Treg; TNFR2⁻Foxp3⁺ Treg; and TNFR2⁺Foxp3⁻ Teff. Data represent the mean ± SEM of at least three experiments with four to six mice per group. ^###p < 0.001, compared with OVA positive control group (Sal/OVA/OVA). The dashed lines denote the percentages of cells expressing the respective markers derived from the saline negative control group (Sal/Sal/Sal).

Figure 7

CD86 expression on lung CD103⁺ dendritic cell (DC) positively correlates with the proportions of TNFR2⁺ regulatory T cells (Treg) and TNFR2⁺Foxp3⁺ that are expanded (Ki67⁺). Naïve mice (n = 5–7 per group per time point) received PS50G i.t on day 0 or saline as control. Samples were collected on days 1, 3, 7, and 30. (A) Stained lung cells were gated on viable MHCII^highCD11c⁺, followed by gating on CD11b⁻CD103⁺ cells on day 3 post instillations. Expression of CD86 population in saline (gray line, filled histogram) and PS50G (black line, open histogram). MFI of MHCII^highCD11c⁺CD11b⁻CD103⁺ DC on day 3 post instillations. Data represent the mean ± SEM. ***p < 0.001 of at least three experiments. The percentages of (B) TNFR2⁺Foxp3⁺ Treg (left panel), TNFR2⁺Foxp3⁺Ki67⁺ Treg (right panel), and (C) TNFR2⁺FoxP3⁻ Treg (left panel) positively correlated with MFI of CD86 on CD103⁺ DC (MHCII⁺CD11c⁺CD11b⁻). The percentages of (C) TNFR2⁻FoxP3⁺ Treg (right panel) negatively correlated with CD103⁺ DC. Open circles correspond to saline group (n = 5–8) and closed circles correspond to PS50G (n = 11) data partially from days 1, 3, 7, and 30.