To distinguish among these two opportunities, we evaluated the impact of plasmin (a hundred nM) on the phagocytosis of protease-resistant223488-57-1 fluorescent microparticles (five hundred nm diameter). Soon after 3 h of co-incubation, plasmin significantly elevated equally the variety of microparticles per cell (Fig 2A) and the number of MoDCs with internalized microparticles (Fig 2B). Consequently, this prophagocytic function of plasmin does not depend on proteolytic degradation of the phagocytic target. Importantly, catalytically inactive plasmin had no influence on microparticle uptake following three h (Fig 2A and 2B). Remedy of MoDCs with a ten-fold reduced concentration of plasmin necrotic cells harbour misfolded proteins and produce surface area-sure plasmin which increases their phagocytosis by human MoDCs. (A) Uninjured and necrotic Jurkat lymphocytes have been stained with ten mg/L 7AAD and ten mg /L Thiazine Purple for fifteen min then subjected to movement cytometry. (B) The Triton-insoluble fractions of uninjured and necrotic Jurkat lymphocytes ended up subjected to SDS-Web page beneath minimizing circumstances and subsequent Coomassie staining. (C) Unhurt and necrotic Jurkat lymphocytes ended up incubated with t-PA and/or plasminogen for 15 min and washed. Complete mobile protein lysates had been prepared and subjected to SDS-Webpage under reducing situations and subsequent immunoblot evaluation. Most of the t-PA employed was in solitary-chain type (70 kDa), even so a little extent of two-chain t-PA was also evident (the band indicated by the asterisk at 35 kDa). (D) PKH67-labelled necrotic Jurkat lymphocytes have been dealt with with the indicated reagents for fifteen min, then washed and incubated with PKH26-labelled human MoDCs. 24 h later, the proportion of double-optimistic (PKH67positive and PKH26positive) MoDCs was assessed by flow cytometry. Data are exhibited as fold-change in double-good MoDCs (indicate s.e.m. n = 3 unbiased experiments). Data was normalized to the team exactly where necrotic cells received no exogenous reagent. p<0.01 and p<0.001 by 1-way ANOVA with Newman-Keuls post-hoc analysis similarly increased microparticle uptake after 3 h (not shown) and 24 h (Fig 2C) of coincubation. Interestingly, the observed plasmin-mediated increase in microparticle uptake was only mildly attenuated by EACA (a lysine analogue) after 24 h (Fig 2C) of co-incubation. This finding suggests that plasmin may not solely rely on kringle-mediated interactions with dendritic cell-surface receptors to enhance phagocytic function.Previous studies have shown that the uptake of microparticles by dendritic cells is an actindependent process, whereas the uptake of nanoparticles (40 nm in diameter) uses cholesterol/ caveolae and clathrin-mediated endocytic mechanisms [17,213]. Accordingly, we assessed whether plasmin increased the endocytic uptake of nanoparticles. 3 h treatment of MoDCs with plasmin failed to increase nanoparticle uptake (Fig 2A and 2B). Hence, the ability of plasmin to promote particle uptake is selective for the actin-dependent pathway of phagocytosis, and not the result of a general enhancement of endocytic capacity.The proteolytic activity of plasmin increases the phagocytosis of microparticles, but not nanoparticles, by MoDCs. MoDCs were treated with 500 nm fluorescent microparticles or 40 nm fluorescent nanoparticles in the presence/absence of 100 nM active or inactive plasmin. 3 h later the relative number of particle-positive MoDCs (Panel A) and internalized particles (Panel B) were assessed by flow cytometry. Data shown are mean standard error (n = 3 independent experiments). Data were normalized to the groups where MoDCs received neither plasmin nor inactive plasmin. p<0.05 by 1-way ANOVA with Newman-Keuls post-hoc analysis. (C) MoDCs were treated with 500 nm fluorescent microparticles in the presence/absence of 10 nM active plasmin and/or 10 mM EACA. The relative number of particle-positive MoDCs was assessed by flow cytometry 24 h later. Data are displayed as mean foldchange in particle positive MoDCs s.e.m. (n = 4 independent experiments). Data was normalized to MoDCs receiving neither plasmin nor EACA. p<0.05, p<0.01 and p<0.001 by 1-way ANOVA with Newman-Keuls post-hoc analysis.We next determined whether plasmin was having a broader influence on dendritic cells by assessing the maturation, cytokine levels, morphology and viability of MoDCs. To assess MoDC maturation status, we measured the extracellular levels of an array of cytokines. These studies showed that plasmin-treatment alone did not alter IL-6 (Fig 3A), IL-10 or IL-12 levels (S1 Fig) suggesting that plasmin does not cause MoDC maturation. In contrast, the maturation of MoDCs via LPS-treatment coincided with significant increases in IL-6 (Fig 3A), IL-10 and IL-12 (S1 Fig). Plasmin-treatment did however trigger a 13-fold elevation in extracellular TGF- levels (Fig 3B) which was seemingly unrelated to the maturation status of MoDCs plasmin-treatment increases TGF- release, but not the maturity or viability of MoDCs. A and B) MoDCs were treated with 10 nM active plasmin or inactive plasmin or 500 ng/mL LPS. After 48 h, the concentration of IL-6 (Panel A) and TGF- (Panel B) in the conditioned media was determined. Data are displayed as mean s.e.m (n = 4 independent experiments). IL-6 data was normalized to untreated MoDCs. p<0.05 and p<0.001 by 1-way ANOVA with Newman-Keuls post-hoc analysis. C) MoDCs were incubated in the presence/absence of 1 nM t-PA+100 nM plasminogen. 24 h later the cell surface expression of CD86 was assessed via flow cytometry. The mean fluorescence intensity of the CD86 signal was normalized to that of untreated MoDCs. Data are displayed as mean s.e.m (n = 3 independent experiments). p<0.05 by 2-tailed Welch's unequal variances t-test. 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. Data was normalized to the values for untreated cultures and detergent-treated cultures. Data are shown as mean s.e. m. (n = 2 for staurosporine-treatment and n = 5 independent experiments for all other groups). p<0.0001 by 1-way ANOVA with Newman-Keuls post-hoc analysis given that LPS-treatment failed to alter TGF- levels (Fig 3B). To further examine whether plasmin affects MoDC maturation, we monitored the expression of several immunomodulatory cell-surface markers. Consistent with the notion that plasmin does not cause dendritic cell maturation, plasmin generation was found to decrease expression of CD86, CD70, CD80, CD274 and HLA-DR in MoDCs (Fig 3C and S1 and S4 Figs. 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. We also noted that plasmin elicited a pronounced change in dendritic cell morphology (S2 Fig). 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). 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 [26]. Importantly, the observed plasmin-mediated alterations in MoDC morphology/function were not the result of cellular toxicity, as no reduction in metabolism (Fig 3D) or perturbation of the plasma membrane (Fig 3E) was detected. Collectively, plasmin produces a discrete influence on dendritic cells: by selectively promoting phagocytosis, increasing TGF- expression, decreasing immunomodulatory receptor expression and altering cell morphology, without causing overt stress or maturation of MoDCs.The observation that plasmin-treatment markedly increased total TGF- levels was intriguing since plasmin is known to proteolytically activate TGF- [27], and because TGF- is a potent immunosuppressant of downstream lymphocyte activation [28,29]. We therefore determined whether plasmin impaired the capacity of MoDCs to mount an adaptive immune response. To this end, mixed lymphocyte reactions were performed using MoDCs and allogeneic leukocytes from 4 independent donors. As shown in Fig 4, plasmin-treated MoDCs had significantly reduced capacity to stimulate a mixed lymphocyte reaction. Hence, plasmin dramatically attenuates the ability of MoDCs to promote an allogeneic immune response.Prior studies show that plasmin can modulate dendritic cell function by cleaving cell-surface Annexin A2, which in turn triggers downstream phospho-ERK1/2 (22). Surprisingly, we were unable to detect cleaved Annexin A2 (S1 Fig), or increased phospho-ERK1/2 (not shown) in MoDCs following plasmin-treatment. 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 panand 340 phospho-specific antibodies). Pair-wise comparison of the microarray data produced a high-confidence list of 31 signalling proteins that were differentially regulated in MoDCs by plasmin (S1 Table). This kinomic dataset was then subjected to Ingenuity Pathway Analysis where the analyst was blinded to both the experimental design and the overall project hypothesis. The top three canonical pathways identified by the Ingenuity Pathway Analysis were collated into a single hypothetical signalling network underlying plasmin-mediated immunomodulation (Fig 5A). These analyses suggest that plasmin-mediated immunomodulation involves altered plasmin-treatment of MoDCs suppresses allogeneic lymphocyte proliferation. MoDCs prepared from four different donors were treated with 100 nM plasmin for 24 h, then washed and incubated with allogeneic PBMCs in triplicate for 4 days. 3H-thymidine incorporation over the last 24 h was taken as a measure of lymphocyte proliferation. Data are shown as mean s.e.m. p<0.05, p<0.01 and p<0.001 by unpaired two-tail Student's t-test platelet-derived growth factor (PDGF) receptor signalling, IL-2 receptor signalling and Fc-receptor signalling. 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). Altogether, our kinomic data suggests that future studies should assess whether PDGF and IL-2 signalling in dendritic cells can be modulated by plasmin activity especially within the context of phagocytosis and immunomodulation.We next determined whether plasmin could also promote the phagocytic function of mouse dendritic cells. To this end, bone marrow-derived mouse dendritic cells (BM-mDCs) were incubated with fluorescent microparticles in the presence or absence of mouse plasmin. After 6 h of co-incubation, the extent of microparticle uptake was assessed within conventional CD11c+ dendritic cells. As shown in S3 Fig, plasmin caused a significant increase in the number of CD11c+ dendritic cells with internalized microparticles. Altogether, our results show that plasmin-treatment increases the phagocytic capacity of both human and mouse dendritic cells in vitro.To explore whether plasmin could similarly modulate dendritic cell function in vivo, fluorescent microparticles were injected intradermally into the base of the tail of wild-type mice in the presence or absence of t-PA and plasminogen (alone or in combination). In addition, mice were co-injected with LPS and microparticles to serve as a positive control for enhanced maturation and migration by dendritic cells. Cells within the draining lymph nodes were collected 24 h after injection, and three major dendritic cell types (conventional, plasmacytoid and Langerhans cells) were stained and assessed for microparticle uptake, maturation status and overall number. Consistent with our in vitro data, plasmin generation increased the in vivo phagocytic capacity of all three dendritic cell populations (Fig 6 top panels). Moreover, this increase in phagocytosis did not result in maturation, as determined by CD86 expression (Fig 6 middle panels). Plasmin generation also caused a significant reduction in the number of conventional dendritic cells (cDCs) entering the draining lymph nodes with similar albeit non-significant trends seen for plasmacytoid dendritic cells (pDCs) and Langerhans cells2843633 (Fig 6 bottom panels). The reason for a plasmin-mediated reduction in dendritic cell migration to the draining lymph nodes may relate to plasmin acting as a chemotaxic agent [26,30], whereby plasmin kinomic screening and prediction of the signalling events that underlie plasmin-mediated immunomodulation. (A) MoDCs were incubated in the presence/absence of 1 nM t-PA and 100 nM plasminogen. 3 h later cultures were lysed and subject to kinomic microarray analysis yielding a short list of 31 signalling proteins that were differentially regulated upon plasmin-treatment (see S1 Table for complete dataset). The short list was subjected to Ingenuity Pathway Analysis to yield three canonical signalling pathways (not shown) for plasmin-mediated immunomodulation, which were manually merged and refined to generate a single putative signalling pathway that underlies plasmin-mediated immunomodulation. Red symbols represent upregulated events.Blue symbols represent downregulated events. (B) The kinomic short list of differentially regulated signalling proteins was batch analysed using the Pathway Interaction Database (PID). Shown are the 5 most significantly perturbed pathways of interest (POI), the differentially regulated proteins within each POI and the degree of statistical significance (p-value computed using the default PID hypergeometric distribution test)discourages the migration of dendritic cells away from the injection site. Injection of either tPA or plasminogen alone failed to alter any of the measured dendritic cell parameters indicating that the observed effects require plasmin formation. As expected, and in contrast to the influence of plasmin, LPS injection caused maturation and reduced microparticle uptake in all dendritic cell subtypes. LPS also increased migration of conventional dendritic cells and intradermal plasmin injection increases the phagocytosis of microparticles by multiple mouse dendritic cell types in vivo, but does not trigger their maturation or migration to the draining lymph nodes. Mice were intradermally injected with microparticles in the presence/absence of 0.02 pmol of t-PA, 0.1 pmol of plasminogen or 10 g of LPS. 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.