Acta Diabetol. doi: 10.1016/j.clim.2007.03.002. Front Immunol. Studies have shown that most cells are in fact capable of carrying out phagocytosis. All authors revised the final manuscript and gave final approval of the version to be published. The observed plasmin-mediated suppression of multiple distinct markers suggests that plasmin proteolyses (or downregulates) an array of cell-surface immunomodulatory receptors, consistent with its ability to maintain an immature phenotype. (2008) 8:579–91. Swanton S, Choh AC, Lee M, Laubach LL, Linderman JK, Czerwinski SA, et al. Buffy coats were obtained from blood donations of healthy donors were conducted in accordance with the Declaration of Helsinki, and kindly provided by the Australian Red Cross Blood Service. View all Since the actuation of B and T cells is reliant on the introduction of H. pylori antigens by dendritic cells, this is of vital significance for the result of the safe reaction. 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. Finally, membrane expression of PS receptors and immune-related molecules in DCs from pediatric patients with T1D was also studied to determine whether the activation of tolerogenic signaling pathways was translated to the DC membrane. Contribution of defective PS recognition and efferocytosis to chronic inflammation and autoimmunity. Table 3. here. Immunol., 28 November 2019 Little is known about the potential of plasmin to alter the unique endocytic potential (including phagocytosis) of dendritic cells. D and E) MoDCs were treated with 100 nM plasmin or 200 nM staurosporine (stauro) for 24 h. Cellular metabolism (Panel C) and plasma membrane integrity (Panel D) were assessed by the MTS and LDH assays respectively. Phosphatidylserine-liposomes promote tolerogenic features on dendritic cells in human type 1 diabetes by apoptotic mimicry. The editor and reviewers' affiliations are the latest provided on their Loop research profiles and may not reflect their situation at the time of review. Efficient t-PA-mediated plasmin formation leads to the subsequent proteolytic degradation of the dead cell corpse [7,8]. SR-F, AV, DP-B, and LG-M performed the experiments. PS-liposomes encapsulating human insulin chains A (PSA-liposomes) and B (PSB-liposomes), were prepared as described (21) with the following physicochemical features: PSA-liposomes were 30 mmol/L of lipid concentration, 783 ± 104 nm of diameter and −38.15 ± 1.20 mV of ζ-potential; PSB-liposomes had 30 mmol/L of lipid concentration, 1040 ± 71 nm of diameter and −38.05 ± 0.07 mV of ζ-potential. Importantly, the expression of tolerogenic markers was also found upregulated in adult DCs after the capture of PSAB-liposomes in our previous RNA-seq analysis (21). As in adults, IL-10 was detected at very low levels in all conditions, correlating with the above-mentioned gene expression results, although this could be explained by the timing of the experiment. The observation that plasmin-treatment markedly increased total TGF-β levels was intriguing since plasmin is known to proteolytically activate TGF-β [27], and because TGF-β is a potent immunosuppressant of downstream lymphocyte activation [28,29]. By exploiting the inherent ability of apoptotic cell clearance to induce tolerance, a synthetic lipid-vesicle strategy that mimics apoptotic beta cells by being enriched in PS and encapsulating insulin was designed (20). CIBER of Diabetes and Associated Metabolic Diseases (CIBERDEM) is an initiative from Instituto de Salud Carlos III (Spain). Culture supernatants from DCs (n≥3 for control subjects and n≥3 for patients with T1D) were collected and frozen at −80°C until use. ICN2 was supported by the Severo Ochoa program from Spanish MINECO (Grant No. Moreover, after liposome capture, the expression levels of genes of PS receptors were preserved or even showed a tendency to increase in comparison to iDCs. doi: 10.1083/jcb.201004096, 7. These signals, called Damage-Associated Molecular Patterns (DAMPs), represent an array of generic motifs that are recognised by a cognate set of humoral factors and peri-cellular receptors which, in turn, instruct the efficient removal of dead cells [1,2]. Trahtemberg U, Mevorach D. Apoptotic cells induced signaling for immune homeostasis in macrophages and dendritic cells. Ways forward for tolerance-inducing cellular therapies- an AFACTT perspective. Overall, these data suggest that several factors, e.g., intrinsic defects, dysglycemia and inflammation, may contribute to the impairment of phagocytosis in T1D. An additional component to the removal of unwanted proteins is the phagocytic arm of the innate immune system. Cell Immunol. It is interesting to note, however, that increasing plasmin formation (via a lowering of the t-PA-inhibitor protein ‘plasminogen activator inhibitor-1’) suppresses myosin-induced autoimmune myocarditis in rats [35]. It would also be interesting to determine whether the capacity of LPS to promote MoDC maturation and/or release of IL-10 or IL-12 could be modulated by plasmin formation. Creusot RJ, Postigo-Fernandez J, Teteloshvili N. Altered function of antigen-presenting cells in type 1 diabetes: a challenge for antigen-specific immunotherapy? 24 h later the cell surface expression of CD86 was assessed via flow cytometry. (2012) 35:2303–10. We would like to thank Ms Chindu Govindaraj for organising the delivery of buffy coats from the Australian Red Cross Blood Bank and Mr Mutsa Madondo, Department of Immunology, Monash University for assistance with the immunostaining of human MoDCs. 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). Is the Subject Area "Plasmins" applicable to this article? Copyright © 2019 Rodriguez-Fernandez, Murillo, Villalba, Perna-Barrull, Cano-Sarabia, Gomez-Muñoz, Aguilera, Maspoch, Vazquez, Bel and Vives-Pi. An attenuated phagocytic activity has also been found in patients with type 2 diabetes, but it was reversed after the improvement of glycemic control (19). Dendritic cells are a vital component of the innate immune system, which constitutes the body's first line of defense against infectious agents and tumor cells. Click through the PLOS taxonomy to find articles in your field. DCs from patients with T1D show impaired phagocytic activity in correlation with the time of disease progression. Yes A minimum of 10,000 events per sample was acquired using FACS LSRFortessa (BD Biosciences). However, other factors, such as IDO and IL-10, have been shown to inhibit T cell activation by DCs after efferocytosis, but we did not observe any increase in IDO1 and IL10 expression, in line with similar studies (39). No, Is the Subject Area "Lymph nodes" applicable to this article? Furthermore, plasmin-treated dendritic cells had an attenuated capacity to trigger allogeneic lymphocyte expansion. Yes Liposome capture was assessed by flow cytometry (FACSCanto II, BD Biosciences). Conversely, defects in dead cell removal underlie many diseases including lupus [3], cystic fibrosis [4], atherosclerosis [5] and bacterial infection [6]. (2017) 33:127–44. Thus, the here presented data are consistent with a biological tolerogenic effect in pediatric DCs after liposome uptake. Liposome-based immunotherapy against autoimmune diseases: therapeutic effect on multiple sclerosis. As expected, phagocytosis was not observed at 4°C (Figure 2A). Quite unexpectedly, here we found that the phagocytic activity of monocyte-derived DCs from pediatric patients with T1D was impaired, and this defect deteriorated with the progression of the disease but did not affect the induction of a tolerogenic phenotype after phagocytosis. (B) The Triton-insoluble fractions of uninjured and necrotic Jurkat lymphocytes were subjected to SDS-PAGE under reducing conditions and subsequent Coomassie staining. Relative quantification was performed by normalizing the expression of each gene to that of the reference gene GAPDH (Hs02758991_g1), as described in the 2−ΔCt method (25). Further characterisation showed that this change in cell morphology was rapid (occurred within 3 h), sensitive (caused by 0.1 nM plasmin), dependent upon proteolytic activity (inhibited by aprotinin), and only partially mediated by lysine-binding (mildly attenuated by EACA) (n = 3; not shown). Altogether, our results show that plasmin-treatment increases the phagocytic capacity of both human and mouse dendritic cells in vitro. MoDCs were incubated in the presence/absence of 1 nM t-PA + 100 nM plasminogen. Dendritic cells are tree-shaped cells found in the skin, intestinal tract, respiratory tract, and lymph nodes. LPS also increased migration of conventional dendritic cells and Langerhans cells to the draining lymph nodes (Fig 6). Unfortunately, T1D incidence is dramatically increasing in children (22, 23), in whom the disease is more aggressive and entails additional management complications. As for the immunoregulatory profile, DCs from pediatric patients with T1D showed higher expression of PPARG and TGFB1—two genes downstream in the efferocytosis signaling pathway—than control subjects. Differences were found between groups (*p < 0.05, **p < 0.01, Mann-Whitney test). (n = 8–11 independent experiments). Interestingly, most of the differences gathered in the first year of evolution were restored in the second year. Whether this alteration is instrumental in the progression of the autoimmune attack toward the clinical manifestation of T1D is currently under investigation. Taken together, our findings support the notion that misfolded proteins formed during necrosis represent a bona fide DAMP that activates plasmin and thereby promotes the proteolytic and phagocytic removal of dead cells. Peripheral blood mononuclear cells (PBMCs) were isolated from buffy coats using Ficoll-Paque density gradient centrifugation according to manufacturer’s instructions. Eizirik DL, Grieco FA. Cells were washed twice with RPMI media +10% FCS and then twice with PBS. Fluorescent liposomes (PSOG488-liposomes) were generated adding lipid-conjugated fluorescent dye Oregon Green 488 (OG488) 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (ThermoFisher Scientific, Waltham, MA, USA) and had a lipid concentration of 30 mmol/L, a diameter size of 836 ± 217 nm (mean±SD), and a ζ-potential of −38.90 ± 2.52 mV. (1999) 26:259–65. doi: 10.2337/diabetes.49.8.1325, 6. Chen C, Cohrs CM, Stertmann J, Bozsak R, Speier S. Human beta cell mass and function in diabetes: Recent advances in knowledge and technologies to understand disease pathogenesis. The plasmin-stimulated dendritic cell, because of its immature status and because of high TGF-β levels is unable to effectively trigger lymphocyte proliferation and adaptive immunity. On the one hand, efferocytosis of apoptotic beta cells was mimicked using previously-described synthetic microvesicles displaying features of apoptotic cells (20). Wrote the paper: RJB ALS MP RLM. doi: 10.1007/s00592-019-01427-1. Files are presented in GraphPad Prism v.6.01 format. Together with an increased phagocytic capacity, plasmin-treated dendritic cells maintain an immature phenotype, exhibit reduced migration to lymph nodes, increase their expression/release of the immunosuppressive cytokine TGF-β, and lose their capacity to mount an allogeneic response. Data are displayed as mean ± s.e.m (n = 3 independent experiments). Confocal micrographs were taken on a Nikon A1r-si resonant scanning confocal system (microscope: Nikon Ti; objective: Apo LWD, 40x magnification, 1.15 numerical aperture, water immersion; sequential excitation: 405 nm, 488 nm and 546 nm laser lines; respective emission filters: 450/50 nm, 525/50 nm and 595/50 nm; photomultiplier tube detectors; acquisition software: NIS elements Advanced Research). Removal of dead cells in the absence of concomitant immune stimulation is essential for tissue homeostasis. DCs were generated as described (21). doi: 10.2337/db12-0397, PubMed Abstract | CrossRef Full Text | Google Scholar, 3. Analyzed the data: RJB ALS AELA AS MF YYK MP RLM. Diabetes. and M.P. Liposomes consisted of 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (sodium salt) (Lipoid, Steinhausen, Switzerland), 1,2-didodecanoyl-sn-glycero-3-phosphocholine (Lipoid), and cholesterol (Sigma-Aldrich). Exclusion criteria were being under immunosuppressive or anti-inflammatory treatment, the presence of other autoimmune diseases, type 2 diabetes, pregnancy, and compromised kidney or liver function. We are well-aware of the limitations of the study. Reagents were from Life Technologies unless indicated otherwise. Elliott MR, Ravichandran KS. Firstly, DCs subsets from peripheral blood were found quantitatively altered during the first year of disease, but recovered in the second year of progression. Lastly necrotic Jurkat cells bound substantially more exogenous t-PA and plasminogen than uninjured Jurkat lymphocytes (Fig 1C). Finally, patients at first year had a decreased percentage of the CD11c−CD123− subset when compared to control subjects, patients at onset and at second year, and their numbers were also lower when compared to control subjects and patients at second year. Raw data files of figures presented in this study. The mean fluorescence intensity of the CD86 signal was normalized to that of untreated MoDCs. However, the cell–cell interaction can also take place at a distance via cytokines . Briefly, IL-6 concentration was similar before (iDCs) and after (tolDCs) PSAB-liposomes uptake in all groups. Heat-inactivated foetal calf serum (FCS; 200 μL) was added and incubated for 1 min. [H3]-thymidine (1 μCi/well) was added and co-cultures were incubated for a further 24 h before [H3]-thymidine incorporation was measured using a scintillation counter (Top Count; Packard Instrument, Meriden, CT). VEGF-targeted therapy: mechanisms of anti-tumour activity. Articles, Icahn School of Medicine at Mount Sinai, United States. Mayer-Davis EJ, Lawrence JM, Dabelea D, Divers J, Isom S, Dolan L, et al. This finding was replicated in human DCs from adult patients with T1D (21). J Autoimmun. doi: 10.1007/s10495-015-1090-8, 2. We therefore determined whether plasmin impaired the capacity of MoDCs to mount an adaptive immune response. *p<0.05, **p<0.01 and ***p<0.001 by 1-way ANOVA with Newman-Keuls post-hoc analysis. As for total DCs, percentages and numbers were decreased in patients at first year when compared to control subjects, although patients at second year recovered a normal percentage. Here we report that plasmin formed on necrotic cells promotes their phagocytosis by human dendritic cells. PLOS ONE promises fair, rigorous peer review, doi: 10.1056/NEJMoa1610187, 24. (2017) 12:1231–42. Consistent with our in vitro data, plasmin generation increased the in vivo phagocytic capacity of all three dendritic cell populations (Fig 6; top panels). 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). Virology. Hence, both extracellular degrading enzymes and phagocytic responses contribute to the removal of dead cells [9,10] and are likely to communicate with each other not only to maximise clearance, but also to minimise self-recognition and maintain tissue homeostasis. C) MoDCs were incubated in the presence/absence of 1 nM t-PA+100 nM plasminogen. Blue symbols represent downregulated events. Hence, this pro-phagocytic function of plasmin does not rely upon proteolytic degradation of the phagocytic target.