Dendritic cell populations with different concentrations of lipid regulate tolerance and immunity in mouse and human liver

Abstract

Background & aims: Immune cells of the liver must be able to recognize and react to pathogens yet remain tolerant to food molecules and other nonpathogens. Dendritic cells (DCs) are believed to contribute to hepatic tolerance. Lipids have been implicated in dysfunction of DCs in cancer. Therefore, we investigated whether high lipid content in liver DCs affects induction of tolerance.

Methods: Mouse and human hepatic nonparenchymal cells were isolated by mechanical and enzymatic digestion. DCs were purified by fluorescence-activated cell sorting or with immunomagnetic beads. DC lipid content was assessed by flow cytometry, immune fluorescence, and electron microscopy and by measuring intracellular component lipids. DC activation was determined from surface phenotype and cytokine profile. DC function was assessed in T-cell, natural killer (NK) cell, and NKT cell coculture assays as well as in vivo.

Results: We observed 2 distinct populations of hepatic DCs in mice and humans based on their lipid content and expression of markers associated with adipogenesis and lipid metabolism. This lipid-based dichotomy in DCs was unique to the liver and specific to DCs compared with other hepatic immune cells. However, rather than mediate tolerance, the liver DC population with high concentrations of lipid was immunogenic in multiple models; they activated T cells, NK cells, and NKT cells. Conversely, liver DCs with low levels of lipid induced regulatory T cells, anergy to cancer, and oral tolerance. The immunogenicity of lipid-rich liver DCs required their secretion of tumor necrosis factor α and was directly related to their high lipid content; blocking DC synthesis of fatty acids or inhibiting adipogenesis (by reducing endoplasmic reticular stress) reduced DC immunogenicity.

Conclusions: Human and mouse hepatic DCs are composed of distinct populations that contain different concentrations of lipid, which regulates immunogenic versus tolerogenic responses in the liver.

Figure 1. Distinct liver DC populations are defined by their endogenous lipid content

(a) Live murine liver NPC were gated and CD11c⁺ cells were analyzed for BODIPY staining. (b) FACS sorted CD11c⁺MHCII⁺ High-DC and Low-DC were spun onto slides and assessed by HCS LipidTOX Red neutral lipid staining. (c) Human liver NPC were co-stained using BODIPY and antibodies reactive with HLA-DR and Lineage markers. (d, e) Murine High- and Low-DC were purified by FACS and tested for expression of (d) C/EBPα, Lipoprotein lipase, and PPAR-γ by PCR (***P<0.001) and (e) for expression of GRP78 (BiP), peIF2α, eIF2α, and β-actin by Western blotting. (f) Representative purified High- and Low-DC were stained with H&E and compared with CD11c⁺MHCII⁺ spleen DC or bone marrow-derived DC.

Figure 2. High-DC exhibit an immunogenic phenotype

(a) High- and Low-DC were analyzed for expression of surface markers. Median fluorescence index (MFI) for High- and Low-DC are indicated below respective histograms. (b) Liver and spleen DC production of cytokines and chemokines was measured in cell culture supernatants. (c) Liver DC populations were also assessed by intra-cellular cytokine staining for production of IL-17α and TNF-α. (d) High- and Low-DC were isolated from the human liver by FACS and cultured alone or with LPS before analysis of cytokines in cell culture supernatant. (e) Mouse liver DC production of IL-6 and TNF-α was measured in cell culture supernatant after selective TLR ligation. (f) TLR expression in High- and Low-DC was determined by flow cytometry. Experiments were repeated 3–4 times using 3 mice per group (*P<0.05; **P<0.01; ***P<0.001).

Figure 3. High-DC potently activate of CD4⁺ T cells whereas Low-DC generate Tregs

(a) CD4⁺OT-II cellular proliferation, (b) surface phenotype, (c) and cytokine production was measured after co-culture with High- or Low-DC loaded with Ova_323–339 peptide or culture with peptide-pulsed spleen DC. (d) CD4⁺OT-II T cell proliferation was also measured in vivo in the draining popliteal lymph node after footpad immunization with saline, or antigen-loaded High- or Low-DC. The fraction and total number of proliferating CD4⁺ T cells is shown. (e) After five days of DC co-culture with allogeneic CD4⁺ T cells, CD4⁺CD25⁺ cells were gated and analyzed for co-expression of Foxp3. The fraction and total number of CD25⁺Foxp3⁺ cells is shown. Experiments were performed in triplicate and repeated three times (*P<0.05; **P<0.01; ***P<0.001).

Figure 4. High-DC generate potent de novo CTL, enhance pre-existing CTL, and mediate tumor protection

(a) Lysis of Ova-expressing targets in the liver and spleen were measured in mice twice immunized with High- or Low-DC.Ova_257-264. (b) Similarly, the fraction of Ova-Tetramer⁺ T cells among all CD8⁺ T cells was measured in the liver of mice immunized using peptide-pulsed High-DC, Low-DC, or bulk spleen DC. (c) Cytokines were measured in day four spleen CTL cultures that were restimulated with Ova_257-264 peptide. (d) CTL lysis of Ova-expressing targets in the spleen as well as (e) IL-2 and IL-10 production in spleen CTL cultures was measured in Rag1 mice that had been reconstituted with OT-I T cells and then immunized with either High- or Low-DC.Ova_257–264. Experimental results were reproduced at least three times (*P<0.05; **P<0.01; ***P<0.001). (f) Mice were challenged with EG7 after immunization with saline or High- or Low-DC or bulk spleen DC loaded with Ova_257-264 peptide. Time to tumor development (P<0.01 for comparison between High- and Low-DC) and mean tumor size at 21 days are shown (mean n=6/group).

Figure 5. Low-DC are poor at capturing antigen and mediate intra-hepatic tolerance

(a) Hepatic DC fluorescence was measured 1h after feeding mice Ovalbumin conjugated to APC. MFIs are indicated. (b) High- and Low-DC uptake of Ovalbumin and Mannosylated-Albumin were measured in vitro at various time points and (c) in vivo at 1h after i.p. administration of the respective fluorescent antigens. (d) Restimulated spleen cultures from mice that received adoptively transferred High- or Low-DC from Ovalbumin-fed or sham-fed donors were interrogated for production of IL-2 and (e) IL-10. (f) The number of intra-hepatic Ova Tetramer⁺ cells was measured at 96h in mice treated with OT-I T cells followed by injection of saline, Ovalbumin, or Ovalbumin and C75. Experiments were repeated at least three times (n=3–5 mice per group; *P<0.05; **P<0.01; ***P<0.001).

Figure 6. High-DC activate innate immunity whereas Low DC induce anergy

(a, b) Liver High-and Low-DC and spleen DC were co-cultured with NK cells and interrogated for production of (a) IL-6 and IFN-γ. (b) NK1.1⁺ cells were gated and analyzed for expression of CD69 on flow cytometry. (c, d) Liver DC-iNKT cells co-cultures were interrogated for production of (c) IL-6, IFN-γ, and TNF-α. (d) In addition, iNKT cells were gated and analyzed for expression of CD25. (e) High- and Low-DC were analyzed for expression of Notch1, Jagged1, and Delta4 by flow cytometry. (f) High- and Low-DC were also analyzed for expression of Jagged1, Delta4, and β-actin by Western blotting; (**P<0.01; ***P<0.001).

Figure 7. High-DC immunogenicity is abrogated by blocking TNF-α or lessening ER stress

(a) CD4⁺OT-II T cells were cultured alone or co-cultured with High- or Low-DC.Ova_323-339 in the presence or absence of an mAb against TNF-α. IFN-γ was measured in cell culture supernatant. (b, c) Similarly, DC-NK co-cultures were performed in the presence of TNF-α blockade before measuring (b) IFN-γ and (c) IL-6. (d) Peptide-pulsed High-DC, alone or with chaperone, were used to stimulate OT-II T cells. TNF-α was measured in cell culture supernatant. Experiments were repeated 2–3 times (*P<0.05; **P<0.01; ***P<0.001).