KEY POINTS
- pDCs follow a multi-step adhesion cascade to home to VAT that changes upon HFD.
- pDCs localize in lymphoid clusters with increased density and activation after HFD.
- Blocking the adhesion cascade prevents weight gain and improves glucose tolerance
Abstract
Plasmacytoid dendritic cells (pDCs) display an increased abundance in visceral adipose tissue (VAT) of humans with obesity. In the current study, we set out to decipher the molecular mechanisms of their recruitment to VAT and the functional relevance of this process. We observed increased pDC numbers in murine blood, liver, spleen, and VAT after feeding a high-fat diet (HFD) for 3 wk when compared with a standard diet. pDCs were enriched in fat-associated lymphoid clusters representing highly specific lymphoid regions within VAT. HFD led to an enlargement of fat-associated lymphoid clusters with an increased density and migratory speed of pDCs as shown by intravital multiphoton microscopy. For their recruitment into VAT, pDCs employed P-selectin with E-selectin and L-selectin being only critical in response to HFD, indicating that the molecular cues underlying pDC trafficking were dependent on the nutritional state. Subsequent recruitment steps required α4β1 and α4β7 integrins and engagement of CCR7. Application of fingolimod (FTY720) abrogated egress of pDCs from VAT, indicating the involvement of sphingosine-1-phosphate in this process. Furthermore, HFD altered pDC functions by promoting their activation and type 1 IFN expression. Blocking pDC infiltration into VAT prevented weight gain and improved glucose tolerance during HFD. In summary, a HFD fundamentally alters pDC biology by promoting their trafficking, retention, and activation in VAT, which in turn seems to regulate metabolism.
Introduction
Obesity is a major risk factor for metabolic diseases and became an enormous health burden in modern societies in the last 30 years (1). Excessive nutritional energy is stored in the white adipose tissue dispersed s.c. throughout the body and between internal organs. It serves as a major lipid reservoir and additionally as a source of endocrine mediators. Especially, visceral adipose tissue (VAT) is recognized to be highly metabolic active in humans (2). In mice, white adipose tissue attached to the uterus of females or epididymis of males is primarily known as VAT. In recent years it has become evident that excessive nutritional intake does not only affect body weight and disturb metabolism, but it also dramatically influences immunological homeostasis (3, 4). In VAT, classical dendritic cells (DCs) (5), invariant NK T cells (6), and γδ T cells (7) decrease with obesity whereas CD8+ T cells (8), innate lymphoid cells (9), and macrophages change in composition and performance. VAT-resident immune cells have a variety of different functions, including the regulation of adipokine expression, clearance of apoptotic cells, and extracellular matrix remodeling (3, 10). The shift of immune cell composition, numbers, and phenotype initiated with obesity finally results in a switch toward a proinflammatory cytokine milieu (11–13) that leads to a systemic increase of different inflammatory mediators, including free fatty acids, TNF-α, IL-1β, and IL-6 (14, 15), which in turn promote the development of insulin insensitivity and metabolic diseases (16).
Within the white adipose tissue, clusters of hematopoietic cells have been identified and named fat-associated lymphoid clusters (FALCs) (17). These non-classical lymphoid clusters have been described in omental, mesenteric, gonadal, and pericardial fat (17, 18). Composed of B1 and B2 cells (19), CD4+ T cells, and CD11b+ myeloid cells (18), FALCs are highly vascularized (18) and, in contrast to lymph nodes (LNs), they are not encapsulated (17). However, FALCs of the omentum contain high endothelial venules (HEVs) (20), a specialized type of postcapillary venule essential for lymphocyte trafficking to LNs. Interestingly, FALCs increase in number and size in response to acute or chronic inflammation of the peritoneal cavity to initiate adaptive immune responses (17, 18, 21).
Recently, plasmacytoid DCs (pDCs) gained attention, as these rare innate immune cells increase in frequency in VAT and liver of obese mice and humans (22–24). In contrast to classical DCs, pDCs have a round morphology and are characterized by their secretory function (25). They are capable to sense viruses and self-nucleic acids (26) and respond with rapid and robust type 1 IFN (IFN-I) expression and secretion (27). Despite few molecular differences, the function, the overall phenotype, and the core gene expression program is conserved between human and murine pDCs (28), and several pDC-specific surface markers have been established. Murine pDCs express sialic acid binding Ig-like lectin H (SiglecH), plasmacytoid dendritic cell Ag-1 (PDCA-1, CD317), Ly6C, and CD45R. In steady state, pDCs express low levels of MHC class II and costimulatory molecules and their expression increases upon activation (29). The generation of pDCs is mainly restricted to the bone marrow (BM), from where they enter the blood and secondary lymphoid organs to continuously patrol through the body. In the LN, they reach T cell areas mainly through HEVs by employing a specific adhesion cascade (30, 31). Under steady-state conditions, pDCs require L-selectin and during inflammation additionally E-selectin to allow rolling in the LN (32). Furthermore, β1 and β2 integrins as well as multiple chemokine receptors such as CCR7 and CCR9 are essential for pDCs to adhere and transmigrate into the LN, gut, and thymus tissue (33, 34). Similar to T cells, pDCs egress from secondary lymphoid organs back into the blood by sphingosine-1-phosphate (S1P)–mediated signaling (35). S1P is a lipid mediator that is present at high concentrations in plasma and lymph and couples to five different G protein–coupled S1P receptors (S1PRs) (36). The decisive role of S1P in immune cell trafficking was discovered when the immunosuppressive agent fingolimod (FTY720) was found to induce S1PR1, S1PR3, S1PR4, and S1PR5 internalization in T cells to render them unresponsive to the S1P gradient toward the circulation trapping them in secondary lymphatic organs (37, 38).
Within the immune system, pDCs are key players in orchestrating sensitization, activation, and differentiation of innate and adaptive immune cells (39). Interestingly, gene expression analyses during obesity show an upregulation of IFN-I genes in VAT (40), and IFN-I receptor-deficient mice fail to develop obesity and insulin insensitivity (24). Furthermore, recent studies suggest that pDCs, which represent a major source of IFN-I, may play a critical role in promoting obesity (41). In this study, we set out to decipher the impact of a high-fat diet (HFD) on the trafficking of pDCs into VAT and their activation within this key region of obesity. We identified the localization of pDCs in VAT and their multistep adhesion cascade facilitating their homing into VAT. HFD resulted not only in increased pDC infiltration and retention, but it also promoted an activated pDC phenotype compared with a standard fat diet (SFD). Within VAT, pDCs accumulated in FALCs where they increased in density and mobility after HFD application. The blockade of pDC infiltration to adipose tissue prevented weight gain and improved glucose tolerance. In summary, we identified a unique trafficking and activation profile of pDCs within VAT in response to HFD that opens up new avenues for treatment of obesity by targeting pDC homing and activation.
Materials and Methods
Animals
C57BL/6 wild-type (WT) mice were purchased from Charles River Laboratories (Wilmington, MA). CCR7−/− mice were bred and housed in the animal facilities of the New Research Building, Harvard Medical School (Boston, MA). The ubiquitin C–tdTomato mice provided by Wolfgang Kastenmüller (MPI Würzburg, Würzburg, Germany) and the SiglecH-GFP reporter mice (42) were bred in the animal facility of the University of Bonn (Bonn, Germany). Homozygous messenger of IFN-β (MOB) mice were obtained from Stefanie Scheu (HHU Düsseldorf, Düsseldorf, Germany) (43). Mice were housed under specific pathogen–free conditions in accordance with National Institutes of Health guidelines. Animal experiments have been approved by the Institutional Review Board and local authorities. Experiments were performed with mice at 6–10 wk of age at Harvard Medical School (Boston, MA), the University of Bonn, the Biomedical Center of the Ludwig-Maximilians-Universität München, and at the Multiphoton Imaging Core Facility at the Walter Brendel Center of Experimental Medicine in Munich, Germany. Normal chow, HFD (Research Diets, open source; D12492), or SFD (Research Diets, open source; D12450J) were applied for 3 wk.
Ab staining and flow cytometry
Unless stated otherwise, all Abs were obtained from BioLegend. The following anti-mouse Abs were used: CD31 (390), SiglecH (551-PE or -FITC), CD45R (RA3-6B2), CD11b (M1/70), CD45 (30-F11), PDCA-1 (927), CD49d (R1-2), CD62L (MEL-14), CXCR3 (CXCR3-176), and CCR7 (4B12). E-selectin and P-selectin binding sites on pDCs were evaluated by staining with E-selectin–human Ig Fc chimera (R&D Systems) and P-selectin–human Ig Fc chimera Ab (R&D Systems). Staining for flow cytometry was performed at 4°C for 20–30 min in FACS buffer (1% FCS in PBS with 2 mM EDTA). Dead cells were excluded by staining with Zombie Aqua (BioLegend, 1:1000 in PBS). Data were acquired by using FACSCanto II (Becton Dickinson) and analyzed with FlowJo software (Becton Dickinson). For in vivo imaging, SiglecH-Alexa 488, CD45-PE, and CD31-Alexa 647 were used.
Statistical analysis
All data are presented as mean ± SEM. Significance was calculated with GraphPad Prism 8. Statistical analyses were performed using a Student t test or one-way ANOVA as indicated. Significance was defined as follows: *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.
In vivo and in vitro expansion of pDCs
Female C57BL/6 mice were injected s.c. with melanoma cell line B16 secreting FLT3L that results in a massive expansion of all DC subsets in vivo (4, 44). Seven days after inoculation, spleen and liver were harvested and pDCs were enriched by gradient centrifugation (Lymphoprep, STEMCELL Technologies) at 800 × g for 20 min at 20°C without a break. pDC content and number were measured by flow cytometry staining before transfer. Cell suspensions were stained with CSFE (Molecular Probes). When indicated, pDCs were generated in vitro by incubating BM with FLT3L for 7 d. Cells were harvested and pDCs were quantified by FACS staining before labeling with a CellTrace Violet cell proliferation kit (Invitrogen).
Pertussis toxin pretreatment
pDCs were treated with pertussis toxin (PTx) (final concentration 200 ng/ml) for 2 h at 37°C and combined with CSFE labeling during the last 20 min before washing with RPMI 1640 containing 2% FCS. Media-treated pDCs were mixed with differentially stained cells and injected (in 200 μl of solution) into the tail veins of mice.
Homing assays
Enriched labeled pDCs were suspended in 2–4 × 107 cells/ml and injected i.v. After 18 h, recipient mice were sacrificed and spleen, inguinal LNs, liver, and VAT were harvested and analyzed by flow cytometry. Homing of pDCs was calculated to the number of injected pDCs. For inhibitory studies, the number of transferred pDCs was measured and pDCs recovered 18 h later in different organs were calculated per 1 million injected pDCs. In competitive homing assays, WT and CCR7−/− cells were either stained with CFSE or with tetramethylrhodamine-5-isothiocyanate (TRITC; Molecular Probes). Switching labeling ensured no effect of toxicities for either cell population. An index for homing was calculated by a ratio of PTx-treated or CCR7−/− versus control cells (100%).
Blocking Abs for inhibition studies
The following mAbs were used: Mel-14 (rat IgG2a, anti-murine L-selectin (45), Bio X Cell] and PS/2 (rat IgG2b, anti-murine α4), which were stored at −70°C in endotoxin-free saline at 1 mg/ml. P-selectin and E-selectin were blocked with RMP-1 and ultra-RME-1/CD62E (BioLegend), respectively. The integrin α4β7 (LPAM) was blocked with DATK32 (BioLegend), CCR9 with blocking Abs (9B1, BioLegend), and CXCR3 with blocking Abs (CXCR3-173, BioLegend). For inhibition studies, 100 μg of Ab was directly injected i.v. before cell transfer (∼1–2 h) or coinjected with pDC suspension. Administration of the S1PR inhibitor FTY720 (1 mg/kg) or PBS was performed as indicated or by daily i.p. injections.
For long-term blockade mice were fed with SFD (D12450J, Research Diets) or HFD (D12492, Research Diets) for 1 wk and injected daily i.p. with 100 µg of mAb against P-selectin (BioLegend) or its isotype control (IgG2a, MOPC-173). Mice were weighed daily and the weight of VAT was analyzed on day 7. Glucose tolerance was tested (i.p. glucose tolerance test [IPGTT]) by injecting 1 mg/kg glucose (Life Technologies) i.p. into 5-h-starved mice. The level of blood glucose was measured at 10-min intervals until 90 min after glucose injection.
