Mechanistic contributions of Kupffer cells and liver sinusoidal endothelial cells in nanoparticle-induced antigen-specific immune tolerance

Abstract

The intravenous delivery of disease-relevant antigens (Ag) by polymeric nanoparticles (NP-Ags) has demonstrated Ag-specific immune tolerance in autoimmune and allergic disorders as well as allogeneic transplant rejection. NP-Ags are observed to distribute to the spleen, which has an established role in the induction of immune tolerance. However, studies have shown that the spleen is dispensable for NP-Ag-induced tolerance, suggesting significant contributions from other immunological sites. Here, we investigated the tolerogenic contributions of Kupffer cells (KCs) and liver sinusoidal endothelial cells (LSECs) to NP-Ag-induced tolerance in a mouse model of multiple sclerosis, experimental autoimmune encephalomyelitis (EAE). Intravenously delivered Ag-conjugated poly(lactide-co-glycolide) NPs (PLG-Ag) distributed largely to the liver, where they associated with both KCs and LSECs. This distribution was accompanied by CD4 T cell accumulation, clonal deletion, and PD-L1 expression by KCs and LSECs. Ex vivo co-cultures of PLG-Ag-treated KCs or LSECs with Ag-specific CD4 T cells resulted in PGE₂ and IL-10 or PGE₂ secretion, respectively. KC depletion and adoptive transfer experiments demonstrated that KCs were sufficient, but not necessary, to mediate PLG-Ag-induced tolerance in EAE. The durability of PLG-Ag-induced tolerance in the absence of KCs may be attributed to the distribution of PLG-Ags to LSECs, which demonstrated similar levels of PD-L1, PGE₂, and T cell stimulatory ability. Collectively, these studies provide mechanistic support for the role of liver KCs and LSECs in Ag-specific tolerance for a biomaterial platform that is currently being evaluated in clinical trials.

Keywords: Immune tolerance; Immunomodulation; Kupffer cells; Liver sinusoidal endothelial cells; Nanoparticles; PLG.

Fig. 1.

PLG nanoparticles distribute to KCs and LSECs in the liver. PLG-Cy5.5 were intravenously injected into C57BL/6 mice (n = 3) over a range of doses from 0 to 2 mg per mouse. (A) At 24 h, the mice were euthanized and PLG-Cy5.5 fluorescence was quantified in the spleens, lungs, and livers by IVIS imaging. (B) The frequency of PLG-Cy5.5 association with KCs and LSECs and median fluorescence intensity was measured by flow cytometry. At each dose in (A), statistical differences between the liver and the lung and spleen were determined using a two-way ANOVA with Tukey’s multiple comparisons test (*p < 0.05, ****p < 0.0001). Comparisons between KCs and LSECs (B) were determined by two-way ANOVA with Sidak’s multiple comparisons test. Error bars indicate SD and data are representative of 2 independent experiments.

Fig. 2.

Intravenous injection of PLG-Ag results in Ag-restricted CD4 T cell accumulation and clonal deletion in the liver. OT-II mice (n = 3) were injected with 2 mg of PLG-OVA (8 μg OVA/mg), or irrelevant PLG-PLP (8 μg PLP/mg). 24 h post-injection, liver nonparenchymal cells were isolated. (A, B) CD4 T cell number per liver and viability were determined by flow cytometry. DAPI exclusion was used to evaluate viability. Statistical differences were determined by unpaired t-test (A) or two-way ANOVA with Tukey’s multiple comparisons test (B). (C) mRNA transcripts for Il10, Ptges2 and Tgfb1 were measured from liver non-parenchymal cells by RT-qPCR. The 2^−ΔΔCT method was used to quantify relative mRNA expression between PLG-OVA- and PLG-PLP-treated mice with 18s-rRNA as an internal reference. Statistical differences were determined by unpaired t-tests. Error bars indicate SD and data are representative of 2 (A, B) and 3 (C) independent experiments.

Fig. 3.

Delivery of PLG-Ag to KCs and LSECs results in Ag-specific upregulation of inhibitory molecule PD-L1. OT-II mice (n = 3) were injected with 2 mg of PLG-OVA or irrelevant PLG-PLP. After 24 h, liver non-parenchymal cells were isolated and analyzed by flow cytometry. (A) Histograms and (B) quantified median fluorescent intensity of costimulatory molecules CD80, CD86, and CD40, and coinhibitory molecule PD-L1 on KCs and LSECs. Statistical differences were determined by individual t-tests. Differences are indicated between PLG-OVA and PLG-PLP. Error bars represent SD and data are representative of 3 independent experiments.

Fig. 4.

KCs and LSECs present particle-derived Ag resulting in moderate T cell expression of CD25 and secretion of immunomodulatory mediators. C57BL/6 mice (n = 3) were injected with 2 mg of PLG-OVA NPs. After 24 h, liver non-parenchymal cells were isolated and KCs and LSECs were sorted by MACS and co-cultured (10⁵ cells) with naïve CD4 OT-II T cells (0.5 × 10⁵ cells). Bone marrow-derived DCs and MØs were used as controls with and without the addition of soluble OVA peptide. (A) The efficiency of Ag presentation was measured by CD25 expression on live CD4 T cells using flow cytometry. (B) Soluble IL-10 and PGE2 were measured in the co-culture supernatants. Statistical differences were determined using one-way ANOVA with Tukey’s multiple comparisons tests. Nonsignificant differences are indicated by matching letters (p > 0.05). Error bars represent SD and data are representative of 5 (A) and 2 (B) independent experiments.

Fig. 5.

KCs are dispensable for PLG-Ag-induced tolerance in EAE. (A) Schematic representation of treatment scheme. On day −21, SJL mice were splenectomized (Splx; n = 7 per condition) or received a sham surgery (n = 5 per condition). After recovery, one splenectomized cohort was injected with 200 μL of 5 mg/mL clodronate liposomes (Clod) to deplete KCs. 24 h later, mice received 2 mg of PLG-PLP or irrelevant PLG-OVA. 7 days following NP treatment (day 0), mice were immunized with PLP/CFA to induce relapsing-remitting EAE. Mice were scored daily by blinded observers. (B) Mice treated with PLG-PLP displayed decreased clinical scores regardless of splenectomy status or KC depletion, demonstrating that KCs are dispensable to PLG-Ag-mediated tolerance in EAE. Statistical differences were determined using the Kruskal-Wallis test (one-way ANOVA nonparametric test) for the course of disease from day 11–33 (p < 0.05). Error bars indicate SEM and data are representative of 2 independent experiments. Splx, splenectomized; Clod, clodronate-treated.

Fig. 6.

Adoptively transferred PLG-Ag-containing KCs are sufficient for inducing tolerance in the EAE model. SJL mice (n = 5) were injected with 2 mg of Cy5.5 labeled PLG-PLP. 24 h later, the liver non-parenchymal cells were isolated, and KCs were sorted based on particle-derived Cy5.5 fluorescence. Particle-containing KCs were adoptively transferred into naïve SJL mice (10⁶ cells per mouse). The mice were immunized 7 days later with PLP/CFA and scored by blinded observers. Statistical differences were determined by the Mann-Whitney test (two-tailed nonparametric test) over the period from day 10–34 (***p < 0.001). Error bars indicate SEM and data are representative of 2 independent experiments.