ORIGINAL RESEARCH published: 28 November 2019 doi: 10.3389/fimmu.2019.02811 Impaired Phagocytosis in Dendritic Cells From Pediatric Patients With Type 1 Diabetes Does Not Hamper Their Tolerogenic Potential Silvia Rodriguez-Fernandez 1 , Marta Murillo 2 , Adrian Villalba 1 , David Perna-Barrull 1 , Mary Cano-Sarabia 3 , Laia Gomez-Muñoz 1 , Eva Aguilera 4 , Daniel Maspoch 3,5 , Federico Vazquez 4 , Joan Bel 2 and Marta Vives-Pi 1,6* 1 Immunology Section, Germans Trias i Pujol Research Institute, Autonomous University of Barcelona, Badalona, Spain, 2 Pediatrics Section, Germans Trias i Pujol University Hospital, Badalona, Spain, 3 Catalan Institute of Nanoscience and Nanotechnology, CSIC and the Barcelona Institute of Science and Technology, Bellaterra, Spain, 4 Endocrinology Section, Germans Trias i Pujol University Hospital, Badalona, Spain, 5 Catalan Institution for Research and Advanced Studies (ICREA), Barcelona, Spain, 6 Biomedical Research Center in Diabetes Network and Associated Metabolic Diseases (CIBERDEM), Edited by: Carlos III Health Institute (ISCiii), Madrid, Spain Jordi Ochando, Icahn School of Medicine at Mount Sinai, United States Type 1 diabetes (T1D) is prompted by defective immunological tolerance, an event Reviewed by: in which dendritic cells (DCs) are crucial as immune response orchestrators. In fact, Daniel Benitez-Ribas, they contribute to maintaining tolerance to self-antigens, but they can also prompt an Hospital Clínic de Barcelona, Spain Giada Mondanelli, immunogenic response against them, leading to autoimmunity. Countless factors can University of Perugia, Italy potentially impact on the proper functionality of the DCs, which range from altered subset *Correspondence: distribution, impaired phagocytic function to abnormal gene expression. Moreover, in Marta Vives-Pi T1D, metabolic dysregulation could impair DC functions as well. Indeed, since T1D mvives@igtp.cat clinical course is likely to be more aggressive in children and adolescents and entails Specialty section: severe dysglycemia, the aim of this study was to analyze circulating DCs subpopulations This article was submitted to in pediatric T1D at different stages, as well as to characterize their phagocytosis ability Immunological Tolerance and Regulation, and tolerance induction potential. Thus, pediatric patients newly diagnosed with T1D, a section of the journal with established disease and control subjects were recruited. Firstly, DCs subsets Frontiers in Immunology from peripheral blood were found quantitatively altered during the first year of disease, Received: 29 August 2019 but recovered in the second year of progression. Secondly, to study the tolerogenic Accepted: 15 November 2019 Published: 28 November 2019 functionality of DCs, liposomes with phosphatidylserine (PS) were designed to mimic Citation: apoptotic beta cells, which are able to induce tolerance, as previously demonstrated Rodriguez-Fernandez S, Murillo M, by our group in DCs from adult patients with T1D. In this study, monocyte-derived Villalba A, Perna-Barrull D, Cano-Sarabia M, Gomez-Muñoz L, DCs from pediatric patients with T1D and control subjects were assessed in terms of Aguilera E, Maspoch D, Vazquez F, PS-liposomes capture kinetics, and transcriptional and phenotypic changes. DCs from Bel J and Vives-Pi M (2019) Impaired pediatric patients with T1D were found to phagocyte PS-liposomes more slowly and less Phagocytosis in Dendritic Cells From Pediatric Patients With Type 1 efficiently than DCs from control subjects, inversely correlating with disease evolution. Diabetes Does Not Hamper Their Nonetheless, the transcription of PS receptors and immunoregulatory genes, cytokine Tolerogenic Potential. Front. Immunol. 10:2811. profile, and membrane expression of immunological markers in DCs was consistent with doi: 10.3389/fimmu.2019.02811 tolerogenic potential after PS-liposomes phagocytosis. In conclusion, T1D progression Frontiers in Immunology | www.frontiersin.org 1 November 2019 | Volume 10 | Article 2811 Rodriguez-Fernandez et al. Dendritic Cells in Pediatric T1D in childhood entails altered peripheral blood DCs subsets, as well as impaired DCs phagocytosis, although tolerance induction could still function optimally. Therefore, this study provides useful data for patient follow-up and stratification in immunotherapy clinical trials. Keywords: dendritic cells, immune tolerance, type 1 diabetes, phagocytosis, autoimmunity INTRODUCTION Interestingly, the gold-standard experimental model of T1D, the non-obese diabetic (NOD) mouse, harbors a deficiency in Type 1 diabetes (T1D) is prompted by defective immunological the clearance of apoptotic cells by macrophages (15). In humans, tolerance to beta cells, resulting in their destruction by polymorphisms in the FCGR2A gene, which encodes for the low- autoreactive lymphocytes. Although the triggering factors affinity IgG Fc region receptor II-a involved in phagocytosis, causing their activation remain elusive, antigen-presenting cells contribute to its pathogenesis (16). However, no data regarding (APCs) are known to contribute to this activation, and so, the phagocytic function of DCs in human T1D are available. to the development of the disease. Among APCs, dendritic In patients with type 2 diabetes, there is growing evidence that cells (DCs), as orchestrators of the immune response, play a impaired phagocytosis in neutrophils (17, 18) and macrophages crucial role in the process. On the one side, they can induce (19) is related to glycemic control, albeit it can be alleviated with and preserve self-tolerance but, on the other side, they can aid improved metabolic regulation. These data suggest that changes in its breach. Indeed, many factors could potentially impact in glucose homeostasis can alter phagocytic processes, with the on the proper functionality of the DCs, ranging from altered particularity that impairment of efferocytosis can contribute to subset distribution, impaired phagocytic function, abnormal the perpetuation of the autoreactivity in T1D. gene expression to defective functionality. As a case in point, By exploiting the inherent ability of apoptotic cell clearance many studies have linked the deficient removal of apoptotic beta to induce tolerance, a synthetic lipid-vesicle strategy that cells performed by phagocytes to the initiation of autoimmunity mimics apoptotic beta cells by being enriched in PS and (1). In fact, beta cells experience massive peaks of apoptosis, not encapsulating insulin was designed (20). This immunotherapy only as a consequence of the autoimmune attack (2, 3), but also in arrested autoimmunity upon administration to NOD mice after physiological islet remodeling during the perinatal period (4, 5). PS-liposomes were phagocyted by DCs, thus eliciting tolerogenic Plasma membrane integrity is maintained during apoptosis, features. This finding was replicated in human DCs from thus preventing the release of immunogenic signals (6). adult patients with T1D (21). Therefore, this strategy achieves This process, accompanied by fast apoptotic cell clearance successful apoptotic mimicry and constitutes a promising by phagocytes—termed efferocytosis—, allows apoptosis to be strategy for tackling autoimmune diseases. immunomodulatory, which is essential in the maintenance of Unfortunately, T1D incidence is dramatically increasing in homeostasis. Apoptotic cell removal is mediated by highly children (22, 23), in whom the disease is more aggressive conserved receptors and ligands, intracellular signaling, and and entails additional management complications. Since severe engulfment mechanisms. One of the most well-characterized dysglycemia could impair DCs functionality, the aim of this study hallmarks of apoptosis is the exposure of phosphatidylserine was to analyze circulating DCs subpopulations in pediatric T1D (PS) on the membrane of apoptotic cells (7). PS interacts with at different stages, as well as to characterize phagocytosis in T1D several PS receptors —e.g., MerTK, CD36, and MFG-E8— on at the onset—hyperglycemic phase— and at established disease— APCs, which promote efferocytosis, and this process confers when glucose levels are better controlled—, and to evaluate the biological advantages, such as peripheral self-tolerance induction role of phagocytosis in tolerance induction. (8, 9). Indeed, efferocytosis prompts immunomodulatory effects in phagocytes, which drive a tolerogenic antigen presentation and anti-inflammatory mediators production (10). However, MATERIALS AND METHODS deficiencies in apoptotic cell removal promote the turning of apoptotic cells into secondary necrotic cells, whereby they Patients and Control Individuals lose their membrane integrity and release danger signals, thus Pediatric patients with T1D (n = 61) were visited as outpatients promoting inflammation and contributing to the initiation of or hospitalized in the Pediatric Section at Germans Trias i Pujol autoimmunity (11, 12). In fact, accumulating evidence points to Hospital. Patients were recruited at the onset and with a longer the relevant role of impaired efferocytosis in the development of evolution (more than 6 months). Pediatric control individuals multiple sclerosis, rheumatoid arthritis and T1D (13, 14). (n = 21) were also recruited. Inclusion criteria were 1–18 years of age and normal body mass index (BMI). Exclusion criteria were being under immunosuppressive or anti-inflammatory treatment, the presence of other autoimmune diseases, type 2 Abbreviations: DCs, Dendritic Cells; iDCs, Immature Dendritic Cells; mDCs, Mature Dendritic Cells; myDCs, Myeloid Dendritic Cells; PBMCs, Peripheral diabetes, pregnancy, and compromised kidney or liver function. Blood Mononuclear Cells; PS, Phosphatidylserine; pDCs, Plasmacytoid Dendritic All the experiments were carried out in strict accordance with Cells; TolDCs, Tolerogenic Dendritic Cells. the principles outlined in the Declaration of Helsinki for human Frontiers in Immunology | www.frontiersin.org 2 November 2019 | Volume 10 | Article 2811 Rodriguez-Fernandez et al. Dendritic Cells in Pediatric T1D research and after the approval of the Committee on the Ethics 783 ± 104 nm of diameter and −38.15 ± 1.20 mV of ζ-potential; of Research of the Germans Trias i Pujol Hospital (PI-16-083). PSB-liposomes had 30 mmol/L of lipid concentration, 1040 ± Signed informed consent was given by the parents of all subjects, 71 nm of diameter and −38.05 ± 0.07 mV of ζ-potential. and directly by children older than 12 years old. Phagocytosis Assay Analysis of Peripheral Blood DC Subsets To determine whether DCs from patients with T1D had a defect In order to analyze DCs subsets, 2 mL blood samples were in phagocytosis in the context of apoptotic cell removal, we collected in EDTA tubes from control subjects and patients tested DCs for their ability to capture synthetic PS-liposomes with T1D at diagnosis, and at first and second year of disease that mimic apoptotic beta cells. Then, DCs from control subjects, progression. Blood samples were lysed (Lysing Buffer, BD recent-onset patients and patients with established disease were Biosciences, San Jose, CA, USA) and stained with antibodies to cultured with 100 µmol/L PSOG488-liposomes at 37◦ C from CD45-AF700, CD3-APCH7, CD19-APCH7, CD14-V450, CD16- 5 min to 6 h. As a negative control, the same assay was performed APC, CD11c-PECy7, CD123-PerCPCy5.5, CD56-PE, HLA-DR- at 4◦ C to confirm that liposomes were captured by phagocytosis. V500, and Slan-FITC (BD Biosciences). A minimum of 10,000 DCs were extensively washed in cold PBS to remove liposomes events per sample was acquired using FACS LSRFortessa attached to the cell membrane. Liposome capture was assessed (BD Biosciences). Percentage and absolute counts (cells/µL) by flow cytometry (FACSCanto II, BD Biosciences). were analyzed using PerfectCount Microspheres (Cytognos SL, Salamanca, Spain) and FACSDiva software (BD Biosciences) Gene Expression Analysis following gating strategies based on international consensus (24). To determine gene expression changes after phagocytosis, DCs were cultured in basal conditions (immature DCs, iDCs) Human DC Generation and with PS-liposomes encapsulating human insulin chains DCs were generated as described (21). Briefly, peripheral blood A (PSA-liposomes) and B (PSB-liposomes), as described (21). (10 mL) was obtained and monocytes were magnetically isolated A mixture of PSA-liposomes and PSB-liposomes (PSAB- using the StraightFrom Whole Blood CD14 MicroBeads kit liposomes, 1 mmol/L) were simultaneously cocultured with (Miltenyi Biotech, Bergisch Gladbach, Germany). Monocytes DCs from control subjects, recent-onset patients and patients were cultured at 106 cells/mL in X-VIVO 15 media (Lonza, with established disease at 37◦ C for 4 h to promote tolerogenic Basel, Switzerland), supplemented with 2% male AB human features (tolerogenic DCs, tolDCs). RNA was isolated (RNeasy serum (Biowest, Nuaillé, France), 100 IU/mL penicillin (Normon Micro Kit, QIAGEN, Hilden, Germany) and reverse-transcribed SA, Madrid, Spain), 100 µg/mL streptomycin (Laboratorio Reig (High Capacity cDNA Reverse Transcription Kit, ThermoFisher Jofré, Sant Joan Despí, Spain), and 1,000 IU/mL IL-4 and Scientific). cDNA synthesis reactions were performed using 1,000 IU/mL GM-CSF (Prospec, Rehovot, Israel). After 6 random hexamers (0.5 mg/mL, BioTools, Valle de Tobalina, days, DCs differentiation yield was assessed by CD11c-APC Madrid, Spain) and reverse transcriptase Moloney-murine- staining (Immunotools, Friesoythe, Germany) and viability was Leukemia-virus (200 U/mL, Promega, Madison, WI, USA). determined by annexin V-PE (Immunotools) and 7-AAD (BD Targeted cDNA was pre-amplified with TaqMan PreAmp Master Biosciences) using FACSCanto II (BD Biosciences). Mix (ThermoFisher Scientific), and qPCR was performed with TaqMan universal assays (ThermoFisher Scientific) Preparation of Liposomes on a LightCycler R 480 (Roche, Mannheim, Germany) Liposomes consisted of 1,2-dioleoyl-sn-glycero-3-phospho- using the following ones: CD36 (Hs00354519_m1), CD68 L-serine (sodium salt) (Lipoid, Steinhausen, Switzerland), (Hs00154355_m1), CD274 (PD-L1) (Hs00204257_m1), 1,2-didodecanoyl-sn-glycero-3-phosphocholine (Lipoid), and IDO1 (Hs00984148_m1), IL10 (Hs00961622_m1), LAIR1 cholesterol (Sigma-Aldrich). Liposomes were prepared using (Hs00253790_m1), MERTK (Hs01031979_m1), MFGE8 the thin-film hydration method from a lipid mixture of (Hs00983890_m1), PDCD1LG2 (PD-L2) (Hs00228839_m1), 1,2-dioleoyl-sn-glycero-3-phospho-L-serine, 1,2-didodecanoyl- PPARG (Hs01115513_m1), TGFB1 (Hs00998133_m1), TNFAIP3 sn-glycero-3-phosphocholine and cholesterol at 1:1:1.33 molar (Hs00234713_m1), TNFSF14 (Hs00542476_g1), and VEGFA ratio, respectively, as described (20). Particle diameter and (Hs00900055_m1). Relative quantification was performed by surface charge (ζ-potential) were measured by dynamic light normalizing the expression of each gene to that of the reference scattering using Malvern Zetasizer (Malvern Instruments, gene GAPDH (Hs02758991_g1), as described in the 2−1Ct Malvern, United Kingdom). Fluorescent liposomes (PSOG488- method (25). liposomes) were generated adding lipid-conjugated fluorescent dye Oregon Green 488 (OG488) 1,2-dihexadecanoyl-sn-glycero- DCs Phenotype After Phagocytosis 3-phosphoethanolamine (ThermoFisher Scientific, Waltham, DCs from patients at onset and with established disease were MA, USA) and had a lipid concentration of 30 mmol/L, a cultured for 24 h with 20 µg/mL human insulin (Sigma-Aldrich, diameter size of 836 ± 217 nm (mean±SD), and a ζ-potential St. Louis, MO, USA) and 1 mmol/L of PSAB-liposomes to of −38.90 ± 2.52 mV. PS-liposomes encapsulating human determine the effect of insulin chains as autoantigens (tolDCs). insulin chains A (PSA-liposomes) and B (PSB-liposomes), were As controls, DCs were either cultured with 20 µg/mL human prepared as described (21) with the following physicochemical insulin (Sigma-Aldrich) to obtain iDCs or adding a cytokine features: PSA-liposomes were 30 mmol/L of lipid concentration, cocktail —TNF-α (1,000 IU/mL, Immunotools), IL-1β (2,000 Frontiers in Immunology | www.frontiersin.org 3 November 2019 | Volume 10 | Article 2811 Rodriguez-Fernandez et al. Dendritic Cells in Pediatric T1D IU/mL, Immunotools), and Prostaglandin E2 (PGE2 , 1 µmol/L, TABLE 1 | Data from the patients with T1D and control subjects included in the Cayman Chemical, Ann Arbor, MI, USA)— for 24 h to obtain peripheral blood DCs subsets analysis. mature DCs (mDCs). Viability and phenotype were analyzed Parameter Control Patients at Patients at Patients at by flow cytometry (FACSCanto II, BD Biosciences). DCs were subjects onset first year second year stained with 7-AAD (BD Biosciences) and antibodies to CD11c- of evolution of evolution APC, CD86-FITC, HLA-ABC-FITC, HLA-DR-FITC, CD14- n 10 11 11 6 PE and CD40-APC (Immunotools), CD36-APCCy7, TIM4- Gender F/M 6/4 (60%/40%) 6/5 (55%/45%) 8/3 (73%/27%) 5/1 (83%/17%) APC, αvβ5 integrin-PE, CD54-PECy7, TLR2-FITC, CXCR4- Age (years) 7.36 ± 2.52 8.35 ± 5.92 10.73 ± 3.73 10.02 ± 2.78 APCCy7, CCR2-APC (BioLegend, San Diego, CA, USA). Data BMI (kg/m2 ) 17.42 ± 2.19 17.19 ± 3.05 17.94 ± 2.59 18.13 ± 2.23 were analyzed using FlowJo software (Tree Star Inc, Ashland, Age at NA 8.35 ± 5.92 10.17 ± 3.70 8.45 ± 2.73 OR, USA). diagnosis (years) Cytokine Production Progression NA 0.00 ± 0.00 0.56 ± 0.18**** 1.57 ± The Human Th1/Th2/Th17 kit (CBA system; BD Biosciences) (years) 0.10****/++++ was used to measure cytokine production. Culture supernatants HbA1c NP 92.05 ± 22.36 55.79 ± 67.21 ± 12.76* from DCs (n≥3 for control subjects and n≥3 for patients (mmol/mol) 10.78**** HbA1c (%) NP 10.57 ± 2.05 7.25 ± 0.99**** 8.30 ± 1.17* with T1D) were collected and frozen at −80◦ C until use. IL- Fasting NP 0.40 ± 0.40 <0.1 in 6/11 <0.1 in 3/6 2, IL-4, IL-6, IFN-γ, TNF-α, IL-17A, and IL-10 were assessed. C-peptide (55%) patients (50%) patients Data were analyzed using CBA software. VEGFA secretion (ng/mL) 0.72 ± 0.90 0.27 ± 0.06 by DCs was determined in culture supernatant by ELISA Stimulated NP 0.98 ± 1.02 NP NP (ThermoFisher Scientific). C-peptide (ng/mL) Statistical Analysis Insulin dose NA 0.70 ± 0.19 0.60 ± 0.24 0.96 ± 0.18*/++ Prism 7.0 software (GraphPad Software Inc, San Diego, CA, (IU/kg/day) USA) was used for the statistical analysis. For comparisons of Data presented as mean ± SD; p value calculated from Mann-Whitney test (*p < 0.05, unpaired data, a non-parametric Mann-Whitney test was used; ****p < 0.0001 when comparing pediatric patients at onset group with pediatric patients at first or second year of evolution; ++ p < 0.01, ++++ p < 0.0001 when comparing pediatric for paired comparisons, the non-parametric Wilcoxon test was patients at first year of evolution with those at second year of evolution). used. ANOVA was used for comparisons with several factors. For F, female; M, male; BMI, Body Mass Index; NA, Not Applicable; HbA1c , glycated correlation between parameters, Spearman’s test was used. A p hemoglobin; NP, Not Performed. value < 0.05 was considered significant. percentage than patients at onset and patients at second year. The RESULTS numbers of pDCs were higher in control subjects when compared to patients at onset and at first year. Finally, patients at first year DCs Subsets From Peripheral Blood Are had a decreased percentage of the CD11c− CD123− subset when Altered at the Onset and Early Stages of compared to control subjects, patients at onset and at second year, T1D Progression and their numbers were also lower when compared to control As a first approach, different subsets of circulating DCs were subjects and patients at second year. Therefore, DCs subsets from assessed in control subjects and patients with T1D at diagnosis, peripheral blood were found quantitatively altered during the and at first year and second year of progression. Their clinical first year of childhood T1D and recovered in the second year data are summarized in Table 1. All subjects met the inclusion of progression. and exclusion criteria and gave informed consent. Aside from differences in the evolution of the disease, patients at onset DC Differentiation Efficiency Is Similar in showed higher glycated hemoglobin (HbA1c ) levels than patients Pediatric Patients and Control Subjects at first year and at second year of progression. Patients at second After observing alterations in DCs subsets in pediatric patients year of progression required higher insulin doses than patients at with T1D, and in order to assess their functionality, DCs onset and at first year of evolution. were derived from peripheral blood monocytes from patients at Thus, four different subsets of DCs in peripheral blood [total diagnosis and with established disease as well as from control DCs, myeloid DCs (myDCs), plasmacytoid DCs (pDCs) and a subjects. Their clinical data are summarized in Table 2. The CD11c− CD123− putative DC subset] were analyzed (Figure 1) BMI of patients with established disease was significantly higher following the gating strategy displayed in Figure S1. As for total than that of control subjects and patients at onset. As expected, DCs, percentages and numbers were decreased in patients at first patients with established disease had a longer evolution and year when compared to control subjects, although patients at significantly lower HbA1c values than patients at onset. Then, second year recovered a normal percentage. The percentage of monocytes were magnetically isolated directly from whole blood, myDCs was increased in patients at first year when compared and differentiated into DCs in vitro. The number of isolated to control subjects, patients at onset and at second year of CD14+ cells (monocytes), viability, and dynamics of the CD11c progression, although no differences were observed in their marker acquisition (differentiation to DCs) at days 1, 4, and 6 of numbers. As for pDCs, patients at first year displayed a higher culture showed similar values in the three groups (Table 3). Frontiers in Immunology | www.frontiersin.org 4 November 2019 | Volume 10 | Article 2811 Rodriguez-Fernandez et al. Dendritic Cells in Pediatric T1D FIGURE 1 | Peripheral blood DC subsets are quantitatively altered at the early stages of T1D. Percentages (left panel) and concentration (cells/µL, right panel) of total DCs (1st row, HLA-DR+ cells of CD3− CD19− CD56− CD14− cells), myeloid DCs (myDCs, 2nd row, CD11c+ CD123− cells of HLA-DR+ cells), plasmacytoid DCs (pDCs, 3rd row, CD11c− CD123+ of HLA-DR+ cells) and the CD11c− CD123− subset (4th row, CD11c− CD123− of HLA-DR+ cells). Data are expressed as mean ± SD; white squares are control subjects (n = 10), white circles are patients at onset (n = 11), black dots are patients at first year of progression (n = 11), and black rhombuses are patients at second year of progression (n = 6). Statistically significant differences were found when comparing the groups (*p < 0.05, **p < 0.01, Mann-Whitney test). DCs From Patients With T1D Display (Figures 2A,B), the phagocytosis rate of PSOG488-liposomes Impaired Phagocytic Activity in Correlation by DCs from patients with established disease was decreased With Disease Progression when compared to control subjects, starting at 15 min of In order to test the ability of DCs to phagocyte lipid vesicles in coculture and throughout the assay. Moreover, DCs from the context of apoptotic cell removal, fluorescent PS-liposomes patients with established disease had phagocyted lower levels of were used in a time-course experiment, coculturing PSOG488- PSOG488-liposomes than patients at onset at 2 h of coculture. liposomes with DCs from each group of subjects. As expected, No significant differences were found when comparing the phagocytosis was not observed at 4◦ C (Figure 2A). At 37◦ C phagocytosis kinetics of DCs from patients at onset and control Frontiers in Immunology | www.frontiersin.org 5 November 2019 | Volume 10 | Article 2811 Rodriguez-Fernandez et al. Dendritic Cells in Pediatric T1D TABLE 2 | Main clinical characteristics and metabolic data of participants previously reported (21). Thus, and as an additional control, we included in the study of phagocytosis and tolerance induction. have included in this study a phagocytosis assay with DCs from Parameter Control Patients Patients with adult patients with established disease, which demonstrates that subjects at onset established disease the impairment of phagocytosis is an inherent feature of pediatric T1D (Figure S2 and Table S1). n 11 14 19 Gender F/M 8/3 (73%/27%) 9/5 (64%/36%) 12/7 (63%/37%) Efferocytosis and Age (years) 9.56 ± 1.66 9.74 ± 3.54 12.13 ± 3.92 Immunoregulation-Related Gene BMI (kg/m2 ) 17.50 ± 2.61 16.57 ± 3.08 20.33 ± 3.80*/++ Expression Is Altered in DCs From Patients Age at diagnosis NA 9.74 ± 3.54 8.46 ± 3.28 (years) With T1D Progression (years) NA 0.00 ± 0.00 3.66 ± 3.00++++ Since the phagocytic capacity of DCs from patients with T1D was (range: 0.5–11.7 years) altered, the gene expression of specific PS receptors (CD36, CD68, HbA1c (mmol/mol) NP 113.50 ± 20.92 61.92 ± 8.75++++ MERTK, MFGE8), and immunoregulatory molecules (PPARG, HbA1c (%) NP 12.54 ± 1.92 7.82 ± 0.80++++ TNFAIP3, TNFSF14, LAIR1, PDL1, PDL2, TFGB1, and VEGFA) Fasting C-peptide NP 0.46 ± 0.28 <0.1 in 13/19 patients was evaluated in DCs before (iDCs) and 4 h after (tolDCs) (ng/mL) (68%) coculture with PSAB-liposomes in the three groups: control 0.4 ± 0.32 subjects, patients at onset and patients with established disease Stimulated NP 0.88 ± 0.58 NP (Figure 3). All these genes were expressed in iDCs. Firstly, no C-peptide (ng/mL) significant differences in the expression of CD36, CD68 nor Insulin dose NA 0.85 ± 0.22 0.89 ± 0.17 MERTK were found between groups, but a significant reduction (IU/kg/day) in MFGE8 expression was detected in DCs from patients with Data presented as mean ± SD. established disease in comparison to the other groups. Moreover, p value calculated from Mann-Whitney test (*p < 0.05 when comparing either group of after liposome capture, the expression levels of genes of PS patients to the control group; ++ p < 0.01, ++++ p < 0.0001 when comparing patients at onset with those with established disease). receptors were preserved or even showed a tendency to increase F, female; M, male; BMI, Body Mass Index; NA, Not Applicable; HbA1c , glycated in comparison to iDCs. In this sense, a tendency for upregulation hemoglobin; NP, Not Performed. of MERTK —a feature of tolDCs (26)— was observed in tolDCs from control subjects (p = 0.06). TABLE 3 | Data from monocyte isolation and DC differentiation. Regarding genes of immunoregulatory molecules, PPARG and LAIR1 expression discerned both stages of T1D, being higher in Parameter Control Patients at Patients with established disease than at the onset and controls. Patients with subjects onset established disease T1D also showed higher levels of TNFAIP3, TNFSF14, PDL1, PDL2, and TGFB1 in DCs than controls. Interestingly, tolDCs n 11 14 19 from patients exhibited a trend for upregulation of the TNFAIP3, Isolated CD14+ cells 2.60 ± 0.84 2.83 ± 1.46 2.57 ± 1.07 (day 1, ×106 cells) TNFSF14, and TGFB1 genes upon PSAB-liposomes capture at Viability (day 1, %) 96.13 ± 2.34 96.29 ± 3.33 97.16 ± 1.87 different stages of the disease. Finally, VEGFA gene expression CD11c purity 4.23 ± 3.24 4.56 ± 2.68 5.41 ± 4.87 did not differ between groups nor after the capture of PSAB- (day 1, %) liposomes. Concerning IDO1 and IL10, a low expression was Differentiation 63.50 ± 18.59 61.46 ± 25.27 71.74 ± 25.79 detected in all conditions (Figure S3). efficiency (CD11c purity, day 4, %) DCs From Pediatric Patients Acquire a Differentiation efficiency (CD11c 92.17 ± 6.06 88.31 ± 9.19 86.95 ± 13.08 Tolerogenic Potential After Phagocytosis purity, day 6, %) of PS-liposomes In terms of cytokine secretion by DCs, the release of IL-6, TNF- Data presented as mean ± SD. No statistical differences were found between groups (Mann-Whitney test). α, IL-10, and VEGFA by DCs from control subjects, recent-onset patients and patients with established disease was consistent with a tolerogenic profile after PSAB-liposomes phagocytosis subjects. To further dissect this finding, the area under the curve (Figure 4). Briefly, IL-6 concentration was similar before (iDCs) (AUC) of the kinetics of capture was calculated (Figure 2C). The and after (tolDCs) PSAB-liposomes uptake in all groups. AUC values of patients with established disease were significantly Moreover, IL-6 secretion increased after maturation stimulus in lower than those of patients at onset and control subjects. DCs (mDCs) from patients with established T1D, and the same Also, patients at onset had significantly lower AUC values than trend was observed in the other groups. Similarly, TNF-α was control subjects. Therefore, the correlation between AUC values not increased after liposome uptake, and its secretion tended and the time of disease progression was analyzed (Figure 2D), to increase in pro-inflammatory conditions. IL-10 concentration discovering that both parameters were significantly and inversely was detected around the limit of detection in all samples. correlated (Spearman’s r = −0.664, p = 0.022). This defect Remarkably, VEGFA concentration was slightly increased in the was only observed in children with T1D, and not in adults, as supernatant of DCs after liposome uptake when compared to that Frontiers in Immunology | www.frontiersin.org 6 November 2019 | Volume 10 | Article 2811 Rodriguez-Fernandez et al. Dendritic Cells in Pediatric T1D FIGURE 2 | DCs from patients with T1D show impaired phagocytic activity in correlation with the time of disease progression. (A) Flow cytometry contour plots and percentages of the capture of PSOG488-liposomes by DCs (CD11c+ ). From left to right, control DCs cultured with PSOG488-liposomes at 4◦ C for 1 h, and DCs from control subjects, patients at onset and patients with established disease cultured with PSOG488-liposomes at 37◦ C for 1 h. One representative experiment of 6 is shown. (B) Time-course of the capture of PSOG488-liposomes by DCs obtained from control subjects (white squares, n = 6), pediatric patients at onset (white circles, n = 6) and with established disease (black dots, n = 6) at 37◦ C. Results are mean±SEM. Comparisons between groups showed significant differences (*p < 0.05, ***p < 0.001, ****p < 0.0001 when comparing control subjects with patients with established disease; ++ p < 0.01 when comparing patients at onset with patients with established disease, Two-way ANOVA). (C) Area under the curve (AUC) of phagocytosis kinetics curves. Differences were found between groups (*p < 0.05, **p < 0.01, Mann-Whitney test). (D) Correlation between AUC values of phagocytosis kinetics curves and disease progression. A significant correlation was found between both parameters (Spearman’s r = −0.664, p = 0.022, Spearman’s correlation analysis). of iDCs in control subjects and patients with established disease 24 h of coculture with PSAB-liposomes (tolDCs), despite the —although patients at onset showed the same tendency (p = phagocytosis defect. First, the viability of DCs from all groups 0.06). IL-2, IL-17A, and IFN-γ were not detected in any condition was higher than 90% in all conditions, thus confirming that PS- of the assay (data below the detection limit). These results, liposomes are not toxic to DCs obtained from pediatric subjects although not always statistically significant, support the ability of (data not shown). No differences were observed in the DCs DCs from children with T1D to achieve a tolerogenic phenotype. phenotype between patients at onset or with established disease, Afterward, we interrogated whether the phenotype of DCs under any of the conditions (Figure 5). Membrane expression of from patients changed toward a tolerogenic profile after PS receptors such as CD36, TIM4 and Integrin αvβ5 was not Frontiers in Immunology | www.frontiersin.org 7 November 2019 | Volume 10 | Article 2811 Rodriguez-Fernandez et al. Dendritic Cells in Pediatric T1D FIGURE 3 | The expression of genes related to efferocytosis and immunoregulation is altered in DCs from patients with T1D. Expression of CD36, CD68, MERTK, MFGE8, PPARG, LAIR1, TNFAIP3, TNFSF14, PDL1, PDL2, TGFB1, and VEGFA genes in immature DCs (iDCs) before and 4 h after (tolDCs) coculture with PSAB-liposomes, analyzed by qPCR. White squares are control subjects (n = 5), white circles are patients at onset (n = 3-5), and black dots are patients with established disease (n = 6). Bars show the mean ± SD. Gene expression signals were normalized to GAPDH expression. No significant differences were found when comparing culture conditions in the same group (Wilcoxon test). Significant differences were found when comparing the same culture conditions in the three groups (+ p < 0.05, ++ p < 0.01, Mann-Whitney test). altered after liposome capture in comparison to iDCs. Then, receptors, TLR2 and CD14, were observed in DCs under the expression of molecules related to the immune function any conditions. of DCs was assessed. Antigen-presenting molecules HLA-ABC displayed a trend for lower expression in tolDCs when compared to mDCs in patients with established disease (p = 0.08); HLA- DISCUSSION DR expression did not exhibit any changes. As for adhesion molecules such as CD54, in patients with established disease, It is well-known that DCs, as orchestrators of the immune tolDCs showed a tendency for higher expression than iDCs (p = response with a crucial role in maintaining self-tolerance, are 0.06). Regarding costimulatory molecules CD40 and CD86, lower highly involved in the pathogenesis of T1D. Several studies expression was observed in tolDCs when compared to mDCs in have enumerated the broad spectrum of alterations that DCs patients with established disease, and patients at onset displayed from these patients have, ranging from genetic alterations, the same pattern. Chemokine receptors CXCR4 and CCR2 to phenotypic and functional divergences (16). Fewer studies, were not upregulated after phagocytosis; however, mDCs from however, have focused on the potential of DCs from adult patients with established disease tended to express higher levels patients to become tolerogenic (27, 28), but no data are available of these molecules when compared to iDCs and tolDCs (p = in pediatric patients. To our knowledge, this is the first work 0.06). Finally, no defects in the expression of pattern recognition that assesses DCs subsets in children with T1D at different Frontiers in Immunology | www.frontiersin.org 8 November 2019 | Volume 10 | Article 2811 Rodriguez-Fernandez et al. Dendritic Cells in Pediatric T1D FIGURE 4 | The cytokine profile after the uptake of PSAB-liposomes by DCs points to tolerogenic potential. Concentration of IL-6, TNF-α, IL-10, and VEGFA secreted by DCs obtained from control subjects (white squares, n ≥ 3), patients with T1D at onset (white circles, n ≥ 3) and patients with established disease (black dots, n ≥ 3). Bars represent immature DCs (iDCs), immature DCs 4 h after the capture of PSA-liposomes and PSB-liposomes (tolDCs), or mature DCs (mDCs) induced with a cytokine cocktail. Data presented as mean ± SD. Significant differences were found when comparing the different conditions in the same group of subjects (*p < 0.05, Wilcoxon test). Differences were not found when comparing the same culture condition in the three groups (Mann-Whitney test). stages of the disease and evaluates their tolerogenic potential. experimental model of the disease, and also induced tolerogenic Indeed, we believe these are paramount aspects to consider features in human DCs from adult patients with T1D (21). in the near future, when antigen-specific immunotherapies In the present study, three similar groups of pediatric will be available and immunomonitoring of the patients will subjects in terms of gender and age were included. Patients be required. with established disease had higher BMI, which was expected Thus, peripheral blood DC subsets were measured to according to the recovery of weight after disease onset and to determine quantitative alterations related to disease progression. the distribution of BMI related to age (32), but nonetheless Interestingly, most of the differences gathered in the first year of healthy, in order to avoid the hypothetical interference of evolution were restored in the second year. Briefly, the percentage inflammatory processes associated with overweight and obesity. of myDCs and pDCs was increased at diagnosis when compared Quite unexpectedly, here we found that the phagocytic activity to the other time points, whereas the CD11c− CD123− subset of monocyte-derived DCs from pediatric patients with T1D was was decreased. In this sense, alterations in myDCs and pDCs impaired, and this defect deteriorated with the progression of subsets have been reported in children with T1D at initial the disease but did not affect the induction of a tolerogenic stages of disease (29–31), and further efforts are warranted phenotype after phagocytosis. This immune dysfunction, not to validate the usefulness of DC subsets as biomarkers in the previously reported in pediatric patients with T1D, agrees with progression of the disease and to provide further insight into their studies that have reported an impaired phagocytic activity in biological significance. macrophages from the NOD mouse, both at early ages—before The alteration in DCs subsets at the early stages of T1D diabetes development—and at adulthood when compared to was an early observation that prompted us to study the other strains (15). An attenuated phagocytic activity has also been tolerogenic functionality of DCs. To do so, we employed a found in patients with type 2 diabetes, but it was reversed after nanotherapy imitating apoptotic beta cells consisting of PS- the improvement of glycemic control (19). Moreover, a reduced liposomes encapsulating insulin (20). These liposomes were capacity for efferocytosis has been reported in other autoimmune phagocyted by murine DCs, thus arresting autoimmunity in the diseases unrelated to glucose metabolism (13). In addition, Frontiers in Immunology | www.frontiersin.org 9 November 2019 | Volume 10 | Article 2811 Rodriguez-Fernandez et al. Dendritic Cells in Pediatric T1D FIGURE 5 | Phagocytosis of PS-liposomes induces a tolerogenic phenotype in DCs from pediatric patients both at onset and established T1D. Membrane expression of CD36, TIM4, Integrin αvβ5, HLA-ABC, HLA-DR, CD54, CD40, CD86, CXCR4, CCR2, TLR2, and CD14 molecules in DCs obtained from patients at onset (white circles, n = 2–3) and with established disease (black dots, n = 4–9). Bars represent immature DCs (iDCs), iDCs after the capture of PSAB-liposomes (tolDCs), and mature DCs (mDCs) induced with a cytokine cocktail, after 24 h. Data are mean ± SD of relative Median of Fluorescence Intensity (MFI), this being MFI of each condition referred to their respective mDCs control. Significant differences were found when comparing culture conditions in the same group (*p < 0.05, **p < 0.01, Wilcoxon test), but not when comparing the same culture conditions in both groups (Mann-Whitney test). Frontiers in Immunology | www.frontiersin.org 10 November 2019 | Volume 10 | Article 2811 Rodriguez-Fernandez et al. Dendritic Cells in Pediatric T1D specific PS receptors are crucial in efferocytosis. As expected, DCs maturation after apoptotic cell removal (40, 41)—, in mice deficient in PS receptors show impaired efferocytosis which tolDCs from the three groups released higher levels of and increased inflammation (11). Overall, these data suggest VEGFA in comparison to iDCs, 4 h after phagocytosis. This that several factors, e.g., intrinsic defects, dysglycemia and further supports that DCs acquire tolerogenic properties after inflammation, may contribute to the impairment of phagocytosis PS-liposomes phagocytosis, even if their phagocytosis ability is in T1D. It is well-known that efferocytosis is not only a impaired. Overall, these results suggest that DCs from patients mechanism of apoptotic cell removal but also a crucial process could mediate immunoregulatory attempts. in the maintenance of immunological homeostasis, and so this Finally, membrane expression of PS receptors and immune- defect could contribute to the persistent lack of self-tolerance. related molecules in DCs from pediatric patients with T1D was Although DCs from pediatric patients with T1D were also studied to determine whether the activation of tolerogenic found to have impaired phagocytosis in correlation with signaling pathways was translated to the DC membrane. These disease progression, DCs from patients at onset and with PS receptors are expressed in iDCs to allow apoptotic cell established disease showed similar expression of PS receptors phagocytosis. As described previously (21), the expression of PS (CD36, CD68, and MERTK), except for MFGE8. Interestingly, receptors was maintained after PSAB-liposomes phagocytosis or DCs from patients with established disease had a decreased even showed a tendency to increase in comparison to iDCs, MFGE8 expression, and this defect could contribute to the thus preserving the ability to efferocyte. As for the expression herein reported phagocytosis impairment. Since several PS of immune-related molecules, tolDCs from pediatric patients, receptors work to internalize apoptotic cells (33), the suboptimal both at onset and with established disease, showed a pattern expression or function of any of them could explain the altered consistent with tolerogenic potential. Indeed, this phenotype phagocytosis. In fact, scarce information has been reported about is similar to that displayed by DCs after PS-liposomes uptake DCs phagocytic activity, since most studies have been conducted in murine models of T1D and multiple sclerosis (20, 42), with macrophages. Thus, it is plausible that DCs may express the and also in human DCs from adult patients with T1D (21). same PS receptors as macrophages but in different proportions, However, the main limitation in the studies of tolDCs is the or express entirely different PS receptors (34), which could be lack of a robust single marker of tolerogenic potential, and accountable for the impaired phagocytosis of DCs. so several parameters that altogether point to the tolerogenic As for the immunoregulatory profile, DCs from pediatric potential of DCs are usually determined. To date, tolDCs are patients with T1D showed higher expression of PPARG and accepted as reluctant to maturation and stable in a state of low TGFB1—two genes downstream in the efferocytosis signaling expression of antigen-presenting and costimulatory molecules, pathway—than control subjects. Moreover, TNFAIP3 displayed along with a tolerogenic-skewed cytokine profile (43). Thus, the higher expression in DCs from patients; interestingly, the here presented data are consistent with a biological tolerogenic expression of Tnfaip3 in murine DCs was reportedly upregulated effect in pediatric DCs after liposome uptake. after efferocytosis (35), thus reducing their rate of apoptotic We are well-aware of the limitations of the study. On the cell clearance and prompting their acquisition of tolerogenic one hand, efferocytosis of apoptotic beta cells was mimicked potential (36). Also, the overexpression of LAIR1—detected in using previously-described synthetic microvesicles displaying DCs from patients with established disease—has been linked features of apoptotic cells (20). However, the engulfment of to an inhibition of cholesterol-particle phagocytosis by murine these liposomes by DCs induces similar effects to those observed macrophages (37). Thus, the expression of these two genes by physiological apoptotic cell phagocytosis and constitutes a combined could also provide a plausible explanation for the good system to test the fitness of DCs. On the other hand, observed impaired phagocytosis of PS-liposomes. In addition, only six PS receptors were explored, and despite their crucial their higher expression in comparison to control subjects, along role in efferocytosis, other receptors could be altered in T1D with that of PPARG, PDL1, PDL2, and TGFB1 genes, could reflect thus impairing DCs phagocytosis. Whether this alteration is a continuous attempt of immunoregulation prompted by the instrumental in the progression of the autoimmune attack toward ongoing autoimmune reaction (38). Importantly, the expression the clinical manifestation of T1D is currently under investigation. of tolerogenic markers was also found upregulated in adult DCs In fact, when the clinical diagnosis of T1D takes place, there after the capture of PSAB-liposomes in our previous RNA-seq is already a significant loss of beta cells in the pancreas of the analysis (21). However, other factors, such as IDO and IL-10, have patient. However, in this scenario, autoimmunity to beta cells been shown to inhibit T cell activation by DCs after efferocytosis, remains active despite the low numbers of alive or functional beta but we did not observe any increase in IDO1 and IL10 expression, cells in the pancreatic islets, and it can destroy these remaining in line with similar studies (39). beta cells. Thus, the altered phagocytic activity observed in Moreover, the results of the characterization of the cytokine DCs could be translated into impaired phagocytosis of actual profile in DCs from pediatric patients were similar to those apoptotic beta cells, which could facilitate their conversion into reported in adults (21), and support the ability of DCs from secondary-necrotic cells if they are not efficiently removed by children with T1D to achieve a tolerogenic phenotype. As in phagocytes. This happening would increase the availability of adults, IL-10 was detected at very low levels in all conditions, danger signals and feed the autoimmune process against beta correlating with the above-mentioned gene expression results, cells. Perhaps, children susceptible to the disease may carry a although this could be explained by the timing of the defect in the efferocytosis ability of phagocytes, which could experiment. Nevertheless, it is worth mentioning the profile further exacerbate immunogenic responses. Interestingly, the of VEGFA secretion—a cytokine involved in the inhibition of here identified defect in the phagocytic activity of DCs from Frontiers in Immunology | www.frontiersin.org 11 November 2019 | Volume 10 | Article 2811 Rodriguez-Fernandez et al. Dendritic Cells in Pediatric T1D pediatric patients with T1D was not detected in adult patients data. MC-S and DM generated the liposomes. SR-F, AV, DP-B, (21), so we can speculate that childhood features (growth, and LG-M performed the experiments. SR-F and MV-P wrote puberty, environmental factors) and diabetes (metabolic control, the manuscript. MM, AV, DP-B, LG-M, and FV reviewed the immune response heterogeneity) could influence DCs behavior manuscript and contributed to the discussion. All authors revised (44, 45). Nevertheless, the preservation of the tolerogenic the final manuscript and gave final approval of the version to potential in DCs from pediatric patients with T1D could help be published. in restraining the autoimmune destruction of beta cells, and in preserving the residual beta cell mass in patients with T1D. FUNDING In conclusion, pediatric peripheral DCs subsets are altered in the first stages of T1D and their phagocytic capacity deteriorates This work has been funded by the Spanish Government with disease progression, although tolerance induction could still (FIS PI15/00198 and PI18/00436) co-financed with function optimally. Despite further studies to draw the intricate the European Regional Development funds (FEDER), picture of this phagocytosis impairment are needed, both the and by DiabetesCero Foundation. CIBER of Diabetes study of peripheral blood DCs and functional DCs studies and Associated Metabolic Diseases (CIBERDEM) is an provide useful data in patient’s follow up, and could aid in the initiative from Instituto de Salud Carlos III (Spain). stratification of patients in immunotherapy clinical trials, since SR-F was supported by the Agency for Management these results could inform about the immunological status of the of University and Research Grants (AGAUR) of the patients’ DCs and their ability to promote a tolerogenic response. Generalitat de Catalunya. ICN2 was supported by the Severo Ochoa program from Spanish MINECO (Grant DATA AVAILABILITY STATEMENT No. SEV-2017-0706). All datasets generated for this study are included in the ACKNOWLEDGMENTS article/Supplementary Material. Special thanks to Ms. N. Real, nurse of the Pediatric Section, ETHICS STATEMENT and to Dr. E. Martinez-Caceres, Dr. A. Teniente and Ms. Bibiana Quirant, from the Immunology Section of the Hospital All the experiments were carried out in strict accordance with Universitari Germans Trias i Pujol, for their help with blood the principles outlined in the Declaration of Helsinki for human samples. We are grateful to Mr. M. Fernandez (IGTP) for his research and after the approval of the Committee on the Ethics of support in flow cytometry and to Ms. D. Culell-Young for Research of the Germans Trias i Pujol Hospital. All subjects gave English grammar assistance. We are indebted to the patients and written informed consent in accordance with the Declaration their families. of Helsinki. SUPPLEMENTARY MATERIAL AUTHOR CONTRIBUTIONS The Supplementary Material for this article can be found SR-F, FV, and MV-P designed the experiments. MM, EA, FV, and online at: https://www.frontiersin.org/articles/10.3389/fimmu. 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Klocperk A, Petruzelkova L, Pavlikova M, Rataj M, Kayserova J, open-access article distributed under the terms of the Creative Commons Attribution Pruhova S, et al. Changes in innate and adaptive immunity over License (CC BY). The use, distribution or reproduction in other forums is permitted, the first year after the onset of type 1 diabetes. Acta Diabetol. provided the original author(s) and the copyright owner(s) are credited and that the (2019). doi: 10.1007/s00592-019-01427-1. [Epub ahead of print]. original publication in this journal is cited, in accordance with accepted academic 32. Swanton S, Choh AC, Lee M, Laubach LL, Linderman JK, Czerwinski SA, practice. No use, distribution or reproduction is permitted which does not comply et al. Body mass index associations between mother and offspring from with these terms. Frontiers in Immunology | www.frontiersin.org 13 November 2019 | Volume 10 | Article 2811