Immunomodulatory effects of nanoparticles on dendritic cells in a model of allergic contact dermatitis: importance of PD-L2 expression

Abstract

Nanoparticle (NP) skin exposure is linked to an increased prevalence of allergic contact dermatitis. In our prior studies using the mouse contact hypersensitivity (CHS) model, we reported that silica 20 nm (SiO₂) NPs suppressed the allergic response and titanium dioxide NPs doped with manganese (mTiO₂) exacerbated it. In this work, we conducted in vitro experiments using bone marrow-derived dendritic cells (BMDCs) to study the combinatorial effect of the potent 2,4-dinitrofluorobenzene (DNFB) hapten sensitizer with SiO₂ and mTiO₂ NPs on BMDC cytotoxicity, cytokine secretion and phenotype using the B7 family ligands. Results show that DNFB and mTiO₂ behave similarly and exhibit proinflammatory characteristics while SiO₂ promotes a naive phenotype. We observe that the B7-H3 (CD276) ligand is only expressed on CD80 + (B7-1) BMDCs. Results from adoptive transfer CHS studies, combined with BMDC phenotype analysis, point to the importance of PD-L2 expression in modulating the adaptive immune response. This work identifies metrics that can be used to predict the effects of NPs on contact allergy and to guide efforts to engineer cell-based therapies to induce hapten specific immune tolerance.

Figure 1

Effects of DNFB, SiO₂ and mTiO₂ exposure on BMDC cytotoxicity as a function of concentration and time. BMDCs were harvested on day 8 and treated with DNFB, SiO₂ and mTiO₂ to study cytotoxicity as a function of concentration and time by flow cytometry. (A) Cell viability as a function of concentration for a 1 h exposure. (B) Cell viability following exposure to the lowest non-cytotoxic concentrations from (A) and exposed over a period of 5 h, 15 h and 24 h. Results indicate that cytotoxicity of DNFB and mTiO₂ NPs on BMDCs was dose- and time- dependent. Live population was normalized the imDC no treatment control. Ordinary one-way ANOVA was performed and compared to imDC. N = 3–5. Mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Figure 2

Effects of DNFB, SiO₂ and mTiO₂ exposure on BMDC cytokine secretion. BMDCs were exposed to a low non-cytotoxic concentrations of each stressor (0.001 mM DNFB, 0.01 mg/mL SiO₂ and 0.005 mg/mL mTiO₂) over a period of 5 h, 15 h and 24 h. ELISA was used to analyze cytokines in cell culture supernatants: (A) IL-6, (B) TNFα, (C) IL-10. All stressors tend to increase IL-6 and TNFα. Only mTiO₂ increased IL-10. Concentration was normalized against % of live cells. Ordinary one-way ANOVA was performed and compared to imDC. N = 3–5. Mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Figure 3

Co-stimulatory molecules from the B7 family quantified by flow cytometry gated on the CD11c + MHCII + subpopulation following BMDC exposure to each stressor over time. The phenotypic characteristics of BMDCs following exposure to a low concentration of each stressor (0.001 mM DNFB, 0.01 mg/mL SiO₂ and 0.005 mg/mL mTiO₂) was followed over time by flow cytometry gated under the CD11c+ MHCII + (Q2.) subpopulation. DNFB and mTiO₂ behave more similar with upregulation of CD86, CD276, and PD-L1 over time. Only DNFB upregulated CD80. Exposure to SiO₂ did not alter these B7 ligands over time. Ordinary one-way ANOVA was performed and compared to imDC. N = 3–5. Mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Figure 4

Co-stimulatory molecules from the B7 family quantified by flow cytometry gated on the CD11c+ MHCII+ and CD80/CD86 subpopulations following BMDC exposure to each stressor over time. Results for DNFB are colored in blue, SiO₂ in green and mTiO₂ in purple. The phenotypic characteristics of BMDCs following exposure to a low concentration of each stressor (0.001 mM DNFB, 0.01 mg/mL SiO₂ and 0.005 mg/mL mTiO₂) was followed over time, 5 h, 15 h, and 24 h, by flow cytometry gated under the CD11c + MHCII + CD80/CD86 subpopulations. Presented here are changes the CD80 + CD86+ double positive (DP) (Q2.) and the CD80-CD86- double negative (DN) (Q4.) subpopulations including the expression levels of PD-L1 (B7-H1), PD-L2 (B7-DC) and CD276 (B7-H3) on the DP and DN subpopulations. For DNFB exposure, the activated DP population clearly increases as the naive DN decreases and PD-L1 expression increased on the DN at 24 h. mTiO₂ exposure over time parallels the effects of DNFB exposure. In contrast, SiO₂ exposure over time tended to decrease the DP subpopulation and it significantly decreased PD-L1 expression in the DP subpopulation. Both SiO₂ and mTiO₂ tended to increased CD276 in the DP at 24 h. The similarities between DNFB and mTiO₂ were evident in CD80/CD86 and PD-L1 expression and distinct from SiO₂. A two-way ANOVA was performed and compared to imDC. N = 3–5. Mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Figure 5

BMDC cytotoxicity and cytokine secretion following co-exposure to DNFB with SiO₂ or mTiO₂ NPs. BMDCs were co-exposed to DNFB plus SiO₂ or mTiO₂ nanoparticles for 24 h. Cell viability and secreted cytokines were measured and compared to DNFB treatment alone and to untreated imDC as a control. (A) SiO₂ NPs were shown to have cytoprotective effect in DNFB co-exposure whereas (B) mTiO₂ NPs induced higher cytotoxicity. Supernatants were collected to analyze cytokine production by ELISA for (C,D) IL-6 which showed co-culture with SiO₂ NPs decreased IL-6 production while co-culture with mTiO₂ did not. (E,F) addition of either NPs did not alter the levels of TNFα produced by DNFB alone and (G,H) the co-culture of DNFB with mTiO₂ NPs increased IL-10 production while SiO₂ NPs did not. Ordinary one-way ANOVA was performed and compared to imDC (*) and DNFB alone (#). Concentration was normalized against % of live cells. N = 3–5. Mean ± SD. */^#p < 0.05, **/^##p < 0.01, ***/^###p < 0.001, ****/^####p < 0.0001.

Figure 6

Co-stimulatory molecules from the B7 family quantified by flow cytometry gated on the CD11c + MHCII + subpopulation following co-exposure of DNFB with NPs. BMDCs were treated with DNFB (0.001 mM DNFB) and co-exposed with SiO₂ (0.01 mg/mL) or mTiO₂ (0.005 mg/mL) NPs for 24 h. Surprisingly, both NPs appeared to suppress the activation of all co-stimulatory molecules relative to DNFB only at 24 h, except for PD-L2, for which mTiO₂ significantly down regulated it. Ordinary one-way ANOVA was performed and compared to imDC (*) and DNFB (#). N = 3–5. Mean ± SD.*/^#p < 0.05, **/^##p < 0.01, ***/^###p < 0.001, ****/^####p < 0.0001.

Figure 7

Co-stimulatory molecules from the B7 family quantified by flow cytometry gated on the CD11c + MHCII + CD80/CD86 subpopulations following 24 h DNFB alone or DNFB plus NP co-exposure. CD80/CD86 subpopulations were divided and CD276 (B7-H3), PD-L1 (B7-H1) and PD-L2 (B7-DC) were gated under, double positive (DP) population (Q2.) or double negative (DN) population (Q4.) to determine whether their expression varies under the different activation states of BMDCs by using the activation markers CD80/CD86 following non-cytotoxic concentrations of (A) 0.001 mM DNFB alone and plus 0.01 mg/mL SiO₂ co-exposure and (B) 0.001 mM DNFB alone and pls 0.005 mg/mL mTiO₂ co-exposure. NPs suppressed BMDCs activation, increased PD-L1 expression and mTiO₂ induced a significant decrease in PD-L2 expression in the DP subpopulation at 24 h while SiO₂ did not. A two-way ANOVA was performed and compared to imDC (*) and DNFB (#). N = 3–5. Mean ± SD. **/^##p < 0.01, ***/^###p < 0.001, ****/^####p < 0.0001.

Figure 8

Comparison of CHS allergic response for different models of sensitization and challenge. (A) Comparison of ear swelling response for DNFB topical (0.05%) vs. S.C. (0.01 mM DNFB) sensitization with 0.2% DNFB challenge on one ear vs. vehicle on the other. No differences between these sensitization methods was observed. (B) Comparison of ear swelling response for S.C. sensitization with DNFB only and challenge with DNFB or DNFB co-exposure with NPs and vehicle on the other. Ear swelling was exacerbated with mTiO₂ NPs but decreased with SiO₂ NPs compared to DNFB alone. Ordinary one-way ANOVA was performed and compared to imDC (*). N = 3–12. Mean ± SD. *p < 0.05, **p < 0.01. (C) Comparison of ear swelling response for S.C. sensitization with BMDC treated 1 h with DNFB only or DNFB + SiO₂ or DNFB + mTiO₂ and challenge with 0.2% DNFB on one ear vs. vehicle on the other. Ear swelling was exacerbated with mTiO₂ but decreased with SiO₂ compared to DNFB alone.