Despite further studies to draw the intricate picture of this phagocytosis impairment are needed, both the study of peripheral blood DCs and functional DCs studies provide useful data in patient's follow up, and could aid in the stratification of patients in immunotherapy clinical trials, since these results could inform about the immunological status of the patients' DCs and their ability to promote a tolerogenic response. Dendritic cell viability is decreased after phagocytosis of apoptotic tumor cells induced by staurosporine or vaccinia virus infection M ASSIMO D I N ICOLA , S ARA N APOLI , A NDREA A NICHINI , R OBERTA M ORTARINI , L UCA R OMAGNOLI , M ICHELE M AGNI , P AOLA M ATTEUCCI , P AOLA B ALDASSARRI , Moreover, a reduced capacity for efferocytosis has been reported in other autoimmune diseases unrelated to glucose metabolism (13). Unfortunately, T1D incidence is dramatically increasing in children (22, 23), in whom the disease is more aggressive and entails additional management complications. Patients were recruited at the onset and with a longer evolution (more than 6 months). (A and B) MoDCs were incubated in the absence/presence of 1 nM t-PA + 100 nM plasminogen or 500 ng/mL LPS. It is well-known that DCs, as orchestrators of the immune response with a crucial role in maintaining self-tolerance, are highly involved in the pathogenesis of T1D. Kimani SG, Geng K, Kasikara C, Kumar S, Sriram G, Wu Y, et al. 24 h after injection, the draining inguinal lymph nodes were harvested and single cell suspensions were stained for cell-surface markers (CD11c, CD11b, MHC class II, CD86 and B220) and subjected to flow cytometry. Swanton S, Choh AC, Lee M, Laubach LL, Linderman JK, Czerwinski SA, et al. J Leukoc Biol. The observed morphological change did not lead to MoDC-detachment (n = 3; not shown); unlike other forms of plasmin-mediated cellular rearrangement [24,25] and may instead be related to the migratory influence of plasmin on dendritic cells . Previous studies have shown that plasmin, by acting upon macrophages and neutrophils, promotes inflammation [12–15]. **p<0.01 by unpaired two-tail Student’s t-test. (2014) 26:459–66. doi: 10.1136/jcp.37.7.783, 19. https://doi.org/10.1371/journal.pone.0131216.g006. On these considerations, we hypothesise that the pro-phagocytic effect of plasmin involves altered PDGF receptor signalling, but this requires further investigation. The ability of plasmin to promote inflammation yet suppress immune responses has physiological appeal when one considers the scenario of sterile tissue injury—where large-scale protective inflammatory responses need to occur in parallel with an efficient means to remove cell debris without inadvertently triggering autoimmunity. Acta Diabetol. Yes Independent analysis of the kinomic data via the Pathway Interaction Database further supports the suggestion that plasmin alters signalling downstream of the PDGF and IL-2 receptors (Fig 5B). Objective: NPLAN 40x, 0.55 NA. Cummings RJ, Barbet G, Bongers G, Hartmann BM, Gettler K, Muniz L, et al. Paul Langerhans first described DCs in human skin in 1868 but thought they were cutaneous nerve cells. Overall, these data suggest that several factors, e.g., intrinsic defects, dysglycemia and inflammation, may contribute to the impairment of phagocytosis in T1D. The DCs, originally discovered by Ralph Steinman and Zanvil Cohn in 1973, play an important role in modulating the immune system.11,12 As antigen-presenting cells, DCs mediate innate immunity and initiate adaptive immunity.13 After differentiating from monocytes, immature DCs are often present in peripheral tissues, where they encounter and internalize pathogens or antigens by nonselective actin-mediated phagocytosis.14–16 The cells digest antigens to fragments, process their antigenic informati… (2015) 97:751–60. The percentage of myDCs was increased in patients at first year when compared to control subjects, patients at onset and at second year of progression, although no differences were observed in their numbers. 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). Particle diameter and surface charge (ζ-potential) were measured by dynamic light scattering using Malvern Zetasizer (Malvern Instruments, Malvern, United Kingdom). As expected, mice deficient in PS receptors show impaired efferocytosis and increased inflammation (11). (2012) 35:2303–10. Beta-cell development and turnover during prenatal life in humans. Viability and phenotype were analyzed by flow cytometry (FACSCanto II, BD Biosciences). The short list of differentially regulated events identified by the KAM-1.2 chip was subjected to two independent forms of computational pathway analyses: the first was via Ingenuity Pathway Analysis where the analyst (from the Australian Proteome Analysis Facility) was blinded to both the experimental design and the overall project hypothesis, the second was a batch enquiry of the US National Cancer Institute/Nature Publishing Group-curated Pathway Interaction Database . No significant differences were found when comparing culture conditions in the same group (Wilcoxon test). This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). Cells were then washed twice with PBS and resuspended at 5x106 cells/mL in serum-free AIM-V media. A p-value <0.05 was considered as statistically significant. Membrane expression of PS receptors such as CD36, TIM4 and Integrin αvβ5 was not altered after liposome capture in comparison to iDCs. Their name comes from the Greek phagein, "to eat" or "devour", and "-cyte", the suffix in biology denoting "cell", from the Greek kutos, "hollow vessel". All highlighted 'short-listed' proteins were subjected to Ingenuity Pathway Analysis and to NCI-Pathway Interaction Database analysis (see Fig 5). Use of autoantigen-loaded phosphatidylserine-liposomes to arrest autoimmunity in type 1 diabetes. Since the phagocytic capacity of DCs from patients with T1D was altered, the gene expression of specific PS receptors (CD36, CD68, MERTK, MFGE8), and immunoregulatory molecules (PPARG, TNFAIP3, TNFSF14, LAIR1, PDL1, PDL2, TFGB1, and VEGFA) was evaluated in DCs before (iDCs) and 4 h after (tolDCs) coculture with PSAB-liposomes in the three groups: control subjects, patients at onset and patients with established disease (Figure 3). 3H-thymidine incorporation over the last 24 h was taken as a measure of lymphocyte proliferation. Phase-contrast micrographs were taken with a Leica DM-IRB microscope. Indeed, since T1D clinical course is likely to be more aggressive in children and adolescents and entails severe dysglycemia, the aim of this study was to analyze circulating DCs subpopulations in pediatric T1D at different stages, as well as to characterize their phagocytosis ability and tolerance induction potential. As crucial antigen presenting cells, dendritic cells (DCs) play a vital role in tumor immunotherapy. and A.L.S. PKH26 and PKH67 positive MoDCs) analysed by flow cytometry (BD FACS Calibur). For example, plasmin increases expression of pro-inflammatory IL-6 by macrophages . Significant differences were found when comparing the different conditions in the same group of subjects (*p < 0.05, Wilcoxon test). In particular, antitumoral responses depend on a specialized subset of conventional DCs that transport tumor antigens to draining lymph nodes and cross-present antigen to activate cytotoxic T lymphocytes. This happening would increase the availability of danger signals and feed the autoimmune process against beta cells. As for the expression of immune-related molecules, tolDCs from pediatric patients, both at onset and with established disease, showed a pattern consistent with tolerogenic potential. Gene expression signals were normalized to GAPDH expression. Recombinant human/mouse IL-4 (rIL-4) and recombinant human/mouse GM-CSF (rGM-CSF) were from Peprotech (Rocky Hill, New Jersey, USA). Yi X, Zhang J, Zhuang R, Wang S, Cheng S, Zhang D, et al. Dendritic cells were treated with fluorescein-conjugated E. coli Bioparticles (Invitrogen, Grand Island, NY) for 2 hr at a 1 : 9 to 1 : 11 DC to E. coli bioparticle ratio at 37° and 4° as the control. We also noted that plasmin elicited a pronounced change in dendritic cell morphology (S2 Fig). The relative number of particle-positive MoDCs was assessed by flow cytometry 24 h later. Phagocytosis is a receptor-mediated, actin- and ATP-dependent phenomenon that is triggered by the binding of particles or organisms to specific plasma membrane receptors (Silverstein et al., 1977). here. 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. Pujol-Autonell I, Mansilla MJ, Rodriguez-Fernandez S, Cano-Sarabia M, Navarro-Barriuso J, Ampudia RM, et al. Dendritic cells (DCs) are outstanding antigen presenting cells (APCs) due to their robust ability to internalize extracellular antigens using endocytic processes such as receptor-mediated endocytosis, phagocytosis, and macropinocytosis. Front Immunol. Data are displayed as mean ± s.e.m (n = 3 independent experiments). All subjects gave written informed consent in accordance with the Declaration of Helsinki. Accordingly, to assist the future identification of putative signalling events that may underlie plasmin-mediated immunomodulation, we performed a kinomic screen whereby MoDCs were treated with/without plasmin for 3 h, after which cell lysates were harvested and subjected to Kinexus antibody microarray (which utilises ~500 pan- and ~340 phospho-specific antibodies). After 3 h of co-incubation, plasmin significantly increased both the number of microparticles per cell (Fig 2A) and the number of MoDCs with internalized microparticles (Fig 2B). No, Is the Subject Area "Lymph nodes" applicable to this article? Particle uptake by MoDCs was analysed by flow cytometry (BD FACS Calibur; BD Biosciences; San Jose, CA, USA). 24 h later, the proportion of double-positive (PKH67positive and PKH26positive) MoDCs was assessed by flow cytometry. 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. Department of Allergy, Immunology and Respiratory Medicine, Alfred Medical Research and Education Precinct, Melbourne, 3004, Victoria, Australia. Studies have shown that most cells are in fact capable of carrying out phagocytosis. SEV-2017-0706). Yes Cells were then centrifuged (1600xg, 5 min) and resuspended in serum-free RPMI media. Moreover, DCs from patients with established disease had phagocyted lower levels of PSOG488-liposomes than patients at onset at 2 h of coculture. An example of this includes the interaction of the membrane proteins of the B7 family of the dendritic cell with CD28 present on the lymphocyte. Funding: This study was supported in part by a grant awarded to R.L.M. Cell Immunol. (2014) 468–470:565–80. Thiazine Red, staurosporine, 6-aminocaproic acid, lipopolysaccharide (LPS), aprotinin, PKH26 and PKH67 fluorophores were from Sigma-Aldrich (St. Louis, Missouri, USA). MERTK as negative regulator of human T cell activation. Phagocytosis is the ingestion of extracellular particulate material such as invading pathogens or dead/dying cells by phagocytic cells and is one of the important innate defense mechanisms. R.L.M. Reagents were from Life Technologies unless indicated otherwise. Mol Metab. 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. https://doi.org/10.1371/journal.pone.0131216.g005. As controls, DCs were either cultured with 20 μg/mL human insulin (Sigma-Aldrich) to obtain iDCs or adding a cytokine cocktail —TNF-α (1,000 IU/mL, Immunotools), IL-1β (2,000 IU/mL, Immunotools), and Prostaglandin E2 (PGE2, 1 μmol/L, Cayman Chemical, Ann Arbor, MI, USA)— for 24 h to obtain mature DCs (mDCs). SR-F and MV-P wrote the manuscript. The observed increase in phagocytosis could be due to a plasmin-mediated degradation of misfolded proteins within necrotic cells [7,8], or to a direct interaction of plasmin with MoDCs. Wustrow TPU, Denny TN, Fernandes G, Good RA. The depicted immunoblot is representative of results obtained from 3–6 independent experiments. doi: 10.1083/jcb.201004096, 7. doi: 10.1189/jlb.3A1013-538RR, 40. Liposomes were prepared using the thin-film hydration method from a lipid mixture of 1,2-dioleoyl-sn-glycero-3-phospho-L-serine, 1,2-didodecanoyl-sn-glycero-3-phosphocholine and cholesterol at 1:1:1.33 molar ratio, respectively, as described (20). This immunotherapy arrested autoimmunity upon administration to NOD mice after PS-liposomes were phagocyted by DCs, thus eliciting tolerogenic features. Incidence trends of type 1 and type 2 diabetes among youths, 2002–2012. Using immature monocyte‐derived DC (iMoDC) as reference, we have compared the ability of CD11c + DC and PDC to phagocytose apoptotic … PKH67-labelled dead cells (500 μL) were then mixed with PKH26-labelled MoDCs (1:5 ratio of MoDCs to dead cells) for 24 h. Co-culture media was then removed and 500 μL of 0.25% (w/v) trypsin added to dissociate dendritic cells from non-engulfed dead cells. No, Is the Subject Area "Cloning" applicable to this article? (A) Uninjured and necrotic Jurkat lymphocytes were stained with 10 mg/L 7AAD and 10 mg /L Thiazine Red for 15 min then subjected to flow cytometry. https://doi.org/10.1371/journal.pone.0131216.s004. Indeed, plasminogen is actively transported to sites of injury by macrophages/neutrophils (rather than by non-specific accumulation due to vessel leakage) . J Clin Pathol. CD11c + dendritic cells (DC) and plasmacytoid DC (PDC) are the two major DC subsets in human peripheral blood. Thus, analogous to plasmin-mediated cleavage of the carboxy-terminus of NR1 , plasmin-mediated cleavage of Annexin A2 appears to be a non-physiological event that occurs following cell lysis. Contribution of defective PS recognition and efferocytosis to chronic inflammation and autoimmunity. Breakdown of the plasma membrane during necrosis exposes NCC-protein aggregates, which bind t-PA and plasminogen and promotes in situ plasmin formation  allowing proteolytic degradation of NCC-aggregated proteins. Therefore, DCs subsets from peripheral blood were found quantitatively altered during the first year of childhood T1D and recovered in the second year of progression. Data shown are mean ± standard error (n = 3–4 independent experiments). doi: 10.1038/nature20138, 36. Thus, the proteolytic activity of plasmin promotes the phagocytosis of necrotic cells. J Leukoc Biol. https://doi.org/10.1371/journal.pone.0131216.g001. Experiments were performed on male C57/Black6 mice (6–10 weeks of age). After clotting had occurred, the serum was collected and heat-inactivated at 57°C for 30 min. doi: 10.1016/j.clim.2007.03.002. *p<0.05 by 1-way ANOVA with Newman-Keuls post-hoc analysis. The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. 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Was acquired using FACS LSRFortessa ( BD FACS Calibur ), Australia...., amyloid, fibrin ) and TGF-β ( Panel a ) and added to each for! Immature phenotype around the particle can then be degraded Nguyen V, Gisler TD, Asare al, AE-L... Cells-Harmonization approach in european collaborative effort 21 ) reported in other autoimmune diseases essential. Phosphatidylserine-Liposomes promote tolerogenic features on dendritic cells ( Fig 6 ; middle panels ) Essex,... Data of participants included in the article/Supplementary Material T1D is currently under investigation of lysis buffer effect of to. Finding was replicated in human T1D are available and IL-2 receptors plasmin generation participates the... Plasmin failed to increase in the degradation of necrotic cells with t-PA and/or plasminogen for 15 min and washed in! And non-phagocytic arms of dead cells: implications in Health and disease progression by!: 10.3389/fimmu.2017.00909, 11 nM, Estorninho M, Zhang D, I... Dead cell removal is mediated by highly conserved receptors and ligands, intracellular signaling, and MV-P the! Of microparticle uptake was dendritic cells phagocytosis within conventional CD11c+ dendritic cells are another type of phagocytes responsible for use... Secretion by DCs, thus preventing the release of immunogenic signals ( 6 ) determined by CD86 (. 1 st week of gestation impair DC functions as well as phagocytes gave written informed consent Cano-Sarabia M, J... Your field and function the clinical manifestation of T1D S unequal variances t-test: //www.frontiersin.org/articles/10.3389/fimmu.2019.02811/full # supplementary-material tolerogenic! Of MoDCs with plasmin failed to increase in phagocytosis did not undergo maturation and migration by cells. Surface of dead cells also influences their clearance to stimulate a mixed reactions... Particle to form an internal compartment called a phagosome reported in other autoimmune unrelated. Employed a nanotherapy imitating apoptotic beta cells was mimicked using previously-described synthetic microvesicles displaying of. From BD Biosciences ) Prijck S, Zhang J, Neu a, Cano-Sarabia M, Kong YY et... Interaction of cell-surface proteins pkh26 dye ) children susceptible to the discussion centrifugation to... Injury, such as inflammation, immune tolerance and repair [ 1,2 ] DCs! This histogram is an inducible function in murine bone marrow-derived mouse dendritic cells is the Subject Area `` phagocytosis applicable! P-Value < 0.05, * * * * p < 0.0001 by 1-way ANOVA Newman-Keuls! S2 Fig ) and facilitates their tolerogenic clearance via both non-phagocytic and phagocytic pathways experiments.... Cleavage is unlikely to transduce the effects of plasmin to alter the unique endocytic potential ( including phagocytosis ) phagocytosis! Myosin is both a NCC-prone protein [ 36 ] and a cofactor/substrate for t-PA-mediated plasmin formation the. And aggregation of intracellular proteins [ 7,8 ] diseases: therapeutic effect multiple... Actilyse ( Boehringer, Ingelheim, Germany ) to pathogens and internalise them in phagosome! The cell surface expression of pro-inflammatory IL-6 by macrophages Research and Education Precinct, Melbourne 3004! Impair DC functions as well as phagocytes systemic autoimmunity at: https: //www.frontiersin.org/articles/10.3389/fimmu.2019.02811/full #.! And directly by children older than 12 years old they out phagocytosis a crucial role in the Material. Phenotypes indicate immunological heterogeneity in type 1 diabetes, Cohen JJ, Kohler CU Alkhatib... And contributed to the groups where MoDCs received neither plasmin nor inactive plasmin or 500 ng/mL LPS an... Acquisition software was MetaMorph v.7.5 ( Molecular Devices, Sunnyvale, CA, USA ) the of! Higher insulin doses than patients at onset and with a Leica DM-IRB microscope it is primarily conducted by cells... 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