Exogenous Thyropin from p41 Invariant Chain Diminishes Cysteine Protease Activity and Affects IL-12 Secretion during Maturation of Human Dendritic Cells

<div><p>Dendritic cells (DC) play a pivotal role as antigen presenting cells (APC) and their maturation is crucial for effectively eliciting an antigen-specific immune response. The p41 splice variant of MHC class II-associated chaperone, called invariant chain p41 Ii, contains an amino acid sequence, the p41 fragment, which is a thyropin-type inhibitor of proteolytic enzymes. The effects of exogenous p41 fragment and related thyropin inhibitors acting on human immune cells have not been reported yet. In this study we demonstrate that exogenous p41 fragment can enter the endocytic pathway of targeted human immature DC. Internalized p41 fragment has contributed to the total amount of the immunogold labelled p41 Ii-specific epitope, as quantified by transmission electron microscopy, in particular in late endocytic compartments with multivesicular morphology where antigen processing and binding to MHC II take place. In cell lysates of treated immature DC, diminished enzymatic activity of cysteine proteases has been confirmed. Internalized exogenous p41 fragment did not affect the perinuclear clustering of acidic cathepsin S-positive vesicles typical of mature DC. p41 fragment is shown to interfere with the nuclear translocation of NF-κB p65 subunit in LPS-stimulated DC. p41 fragment is also shown to reduce the secretion of interleukin-12 (IL-12/p70) during the subsequent maturation of treated DC. The inhibition of proteolytic activity of lysosomal cysteine proteases in immature DC and the diminished capability of DC to produce IL-12 upon their subsequent maturation support the immunomodulatory potential of the examined thyropin from p41 Ii.</p></div

Effect of internalized p41 fragment on the proteolytic activity of cysteine proteases (A) and the secretion of IL-12/p70 (B, C).

<p>Samples (A): cell lysates of non-treated immature DC, immature DC after a 6-h incubation with p41 fragment (0.035 μM, 0.35 μM and 3.5 μM) and non-treated immature DC with 10 μM E-64. Fluorogenic substrate in buffer with DTT was used as blank (BLK). Representative measurements, each of three biological repetitions, are shown. Samples (B and C): cell free supernatants (culture media) of immature DC, preincubated with p41 fragment (0.035 μM, 0.35 μM and 3.5 μM) for 6 h prior to their maturation with TNF-α (B) or LPS (C). Non-treated cells (no preincubation with p41 fragment): immature DC, cultured in the presence of GM-CSF for three days (no maturation), DC matured with TNF-α, and DC matured with LPS. Pretreated but non-matured cells: immature DC, pretreated with 3.5 μM p41 fragment, and cultured in the presence of GM-CSF. IL-12 concentrations (in pg/ml) were measured in triplicate, average values ± SD are shown. (D) Immunolabelled cathepsins L and S in DC lysates. Samples (50 μg per well): (1) immature DC, (2) DC pretreated with 3.5 μM p41 fragment for 6 h, no LPS, (3) DC matured with LPS for 24 h, (4) DC pretreated with 3.5 μM p41 fragment for 6 h and matured with LPS for 24 h.</p

Colocalization of endogenous p41 Ii and lysosomal cysteine cathepsins S, L and H.

<p>Confocal images show double immunofluorescence and localization of labelled endogenous p41 Ii, cathepsin H, cathepsin S, cathepsin L, CD68, LAMP-2 and HLA-DM in immature DC (A, C) and in mature DC after a 3-day maturation with TNF-α (B, D, E). Fluorescence intensities (A, B) are presented with a rainbow color palette (blue–the lowest intensity, red–the highest intensity). Only merged images are shown elsewhere (C, D, E).</p

Localization of immunogold labelled p41 Ii (A–B, E–F) and cathepsin S (C–D).

<p>TEM micrographs: immature DC (A, C), mature DC after a 3-day maturation with TNF-α (B, D) and immature DC treated with inhibitory p41 fragment (3.5 μM) for 6 h (E, F). Membranes appear white (non-contrasted). Bars: 200 nm (A–B, D, E–F) and 100 nm (C).</p

Translocation of NF-κB subunit p65 in immature DC treated with p41 fragment.

<p>Confocal images show localization of immunolabelled p65 in differentiated MUTZ-3 cells: (A) non-stimulated, (B) stimulated with LPS, (C) pretreated with 10 μM SN50 and stimulated with LPS, (D) pretreated with 10 μM SN50M and stimulated with LPS, (E) pretreated with 3.5 μM p41 fragment, (F) pretreated with pretreated with 3.5 μM p41 fragment and stimulated with LPS. Nuclei were stained with DAPI. Bars: 10 μm.</p

Content (A–F) and localization (G–V) of endogenous p41 Ii and MHC II during maturation of DC with TNF-α.

<p>Continuous-line histograms describe the binding of anti-p41 Ii mAb to endogenous p41 Ii. Shadowed histograms represent the corresponding negative controls. Each M1 interval excludes 95% of cells from the corresponding negative control. The percentage of p41 Ii-positive cells in a particular M1 interval and their mean fluorescence intensity (MFI) are stated. A representative analysis of three independent biological repetitions is shown. Confocal images: (H–N) endogenous p41 Ii, (P–V) MHC II (HLA-DR). Bars: 15 μm.</p

Active site titration of cathepsin L (2.5 nM) with inhibitory p41 fragment (0.5 nM to 3 nM).

<p>Released fluorescence was measured in duplicate, average values ± SD are shown. (A) Released fluorescence (dF/dt) is related to p41 fragment/cathepsin L molar ratio. (B) SDS-PAGE and (C) IEF of isolated inhibitory p41 fragment (both stained with silver). ST–standards.</p

Poly(d,l-lactide-co-glycolide)/hydroxyapatite core–shell nanospheres. Part 3: Properties of hydroxyapatite nano-rods and investigation of a distribution of the drug within the composite

A step-by-step analysis of the formation and the drug loading of the poly(d,l-lactide-co-glycolide)/hydroxyapatite (PLGA/HAp) composite was carried out in a perspective of the following parameters: the structure, the morphology and the adsorption/desorption properties of the composite's bioceramic part. The authors demonstrated the importance of the material's capacity to form a fine dispersion of solid HAp particles, as an initial step, for the further loading of the drug and for the formation of the core–shell structures. The nanometer-sized rods of HAp have the capacity of ensuring a rapid adsorption and a controlled desorption of the drug from their surface, and they can act as a nucleating site for the formation of polymeric cores. Each component of this material was labeled with fluorescence dye, which enabled an insight into the distribution of the components in the core–shells that were obtained as the final outcome. Such an analysis showed a high level of uniformity among the cores enclosed within polymeric shells. From a practical perspective, the labeling of each component of the composite can be regarded as an additional functionality of the material: labeling can enable us to monitor its action during the healing process. This ability to be easily detected is expected to enhance the procedure for the controlled delivery of antibiotics after their local implantation of carriers loaded with the antibiotic and to provide more careful control over this process

Salivary Tick Cystatin OmC2 Targets Lysosomal Cathepsins S and C in Human Dendritic Cells

To ensure successful feeding tick saliva contains a number of inhibitory proteins that interfere with the host immune response and help to create a permissive environment for pathogen transmission. Among the potential targets of the salivary cystatins are two host cysteine proteases, cathepsin S, which is essential for antigen- and invariant chain-processing, and cathepsin C (dipeptidyl peptidase 1, DPP1), which plays a critical role in processing and activation of the granule serine proteases. Here, the effect of salivary cystatin OmC2 from Ornithodoros moubata was studied using differentiated MUTZ-3 cells as a model of immature dendritic cells of the host skin. Following internalization, cystatin OmC2 was initially found to inhibit the activity of several cysteine cathepsins, as indicated by the decreased rates of degradation of fluorogenic peptide substrates. To identify targets, affinity chromatography was used to isolate His-tagged cystatin OmC2 together with the bound proteins from MUTZ-3 cells. Cathepsins S and C were identified in these complexes by mass spectrometry and confirmed by immunoblotting. Furthermore, reduced increase in the surface expression of MHC II and CD86, which are associated with the maturation of dendritic cells, was observed. In contrast, human inhibitor cystatin C, which is normally expressed and secreted by dendritic cells, did not affect the expression of CD86. It is proposed that internalization of salivary cystatin OmC2 by the host dendritic cells targets cathepsins S and C, thereby affecting their maturation