Regulation of TGF-ß1-driven differentiation of human lung fibroblasts: emerging roles of cathepsin B and cystatin C.

Lung matrix homeostasis partly depends on the fine regulation of proteolytic activities. We examined the expression of human cysteine cathepsins (Cats) and their relative contribution to TGF-ß1-induced fibroblast differentiation into myofibroblasts. Assays were conducted using both primary fibroblasts obtained from patients with idiopathic pulmonary fibrosis and human lung CCD-19Lu fibroblasts. Pharmacological inhibition and genetic silencing of Cat B diminished α-smooth muscle actin expression, delayed fibroblast differentiation, and led to an accumulation of intracellular 50-kDa TGF-ß1. Moreover, the addition of Cat B generated a 25-kDa mature form of TGF-ß1 in Cat B siRNA-pretreated lysates. Inhibition of Cat B decreased Smad 2/3 phosphorylation but had no effect on p38 MAPK and JNK phosphorylation, indicating that Cat B mostly disturbs TGF-ß1-driven canonical Smad signaling pathway. Although mRNA expression of cystatin C was stable, its secretion, which was inhibited by brefeldin A, increased during TGF-ß1-induced differentiation of idiopathic pulmonary fibrosis and CCD-19Lu fibroblasts. In addition, cystatin C participated in the control of extracellular Cats, because its gene silencing restored their proteolytic activities. These data support the notion that Cat B participates in lung myofibrogenesis as suggested for stellate cells during liver fibrosis. Moreover, we propose that TGF-ß1 promotes fibrosis by driving the effective cystatin C-dependent inhibition of extracellular matrix-degrading Cats.

Long-term maintenance of skin immune system in a NOD-Scid IL2rγ(null) mouse model transplanted with human skin.

We developed a NOD-Scid IL2rγ(null) mouse model transplanted with human skin that brings fundamental insight on in vivo cellular mechanisms of intradermal immunization and antigen presentation by dermal dendritic and epidermal Langerhans cells for skin T-cell immunity. Indeed, T-cell immunity is a crucial checkpoint for the induction of in vivo rapid control of skin infection. With the long-term preservation of a complete human skin immune system, this model offers the unique opportunity not only to better understand mechanisms of skin immune response but also to test new compounds and devices for cutaneous routes of vaccination, as well as new therapeutics approach for skin diseases, allergies or infections.

Targeting MICA/B with cytotoxic therapeutic antibodies leads to tumor control.

Background: MICA and MICB are tightly regulated stress-induced proteins that trigger the immune system by binding to the activating receptor NKG2D on cytotoxic lymphocytes. MICA and MICB are highly polymorphic molecules with prevalent expression on several types of solid tumors and limited expression in normal/healthy tissues, making them attractive targets for therapeutic intervention. Methods: We have generated a series of anti-MICA and MICB cross-reactive antibodies with the unique feature of binding to the most prevalent isoforms of both these molecules. Results: The anti-MICA and MICB antibody MICAB1, a human IgG1 Fc-engineered monoclonal antibody (mAb), displayed potent antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP) of MICA/B-expressing tumor cells in vitro. However, it showed insufficient efficiency against solid tumors in vivo, which prompted the development of antibody-drug conjugates (ADC). Indeed, optimal tumor control was achieved with MICAB1-ADC format in several solid tumor models, including patient-derived xenografts (PDX) and carcinogen-induced tumors in immunocompetent MICAgen transgenic mice. Conclusions: These data indicate that MICA and MICB are promising targets for cytotoxic immunotherapy.

Imaging of extracellular cathepsin S activity by a selective near infrared fluorescence substrate-based probe.

We designed a near-infrared fluorescent substrate-based probe (SBP), termed MG101, for monitoring extracellular cathepsin S (CatS) activity. We conceived a fused peptide hairpin loop-structure, combining a CatS recognition domain, an electrostatic zipper (with complementary charges of a polyanionic (D-Glu)5 segment and a polycationic (D-Arg)5 motif, as well as a N and C terminal Förster resonance energy transfer pair (donor: AlexaFluor680; quencher: BHQ3) to facilitate activity-dependent imaging. MG101 showed excellent stability since no fluorescence release corresponding to a self-dequenching was observed in the presence of either 2 M NaCl or after incubation at a broad range of pH (2.2-8.2). Cathepsins B, D, G, H, and K, neutrophil elastase and proteinase 3 did not cleave MG101, while CatS, and to a lesser extent CatL, hydrolysed MG101 at pH 5.5. However MG101 was fully selective for CatS at pH 7.4 (kcat/Km = 140,000 M-1 s-1) and sensitive to low concentration of CatS (<1 nM). The selectivity of MG101 was successfully endorsed ex vivo, as it was hydrolysed in cell lysates derived from wild-type but not knockout CatS murine spleen. Furthermore, application of the SBP probe with confocal microscopy confirmed the secretion of active CatS from THP-1 macrophages, which could be abrogated by pharmacological CatS inhibitors. Taken together, present data highlight MG101 as a novel near-infrared fluorescent SBP for the visualization of extracellular active CatS from macrophages and other cell types.

Substrate-derived triazolo- and azapeptides as inhibitors of cathepsins K and S.

Cathepsin (Cat) K is a critical bone-resorbing protease and is a relevant target for the treatment of osteoporosis and bone metastasis, while CatS is an attractive target for drugs in autoimmune diseases (e.g. rheumatoid arthritis), emphysema or neuropathic pain. Despite major achievements, current pharmacological inhibitors are still lacking in safety and may have damaging side effects. A promising strategy for developing safer reversible and competitive inhibitors as new lead compounds could be to insert non-cleavable bonds at the scissile P1-P1' position of selective substrates of CatS and CatK. Accordingly, we introduced a 1,4-disubstituted 1,2,3-triazole heterocycle that mimics most of the features of a trans-amide bond, or we incorporated a semicarbazide bond (azaGly residue) by replacing the α-carbon of the glycyl residue at P1 by a nitrogen atom. AzaGly-containing peptidomimetics inhibited powerfully their respective target proteases in the nM range, while triazolopeptides were weaker inhibitors (Ki in the µM range). The selectivity of the azaGly CatS inhibitor (1b) was confirmed by using spleen lysates from wild-type vs CatS-deficient mice. Alternatively, the azaGly bradykinin-derived CatK inhibitor (2b) potently inhibited CatK (Ki = 9 nM) and impaired its kininase activity in vitro. Molecular modeling studies support that the semicarbazide bond of 2b is more favorable than the 1,2,3-triazole linkage of the bradykinin-derived pseudopeptide 2a to preserve an effective affinity towards CatK, its protease target.

The Unusual Resistance of Avian Defensin AvBD7 to Proteolytic Enzymes Preserves Its Antibacterial Activity.

Defensins are frontline peptides of mucosal immunity in the animal kingdom, including birds. Their resistance to proteolysis and their ensuing ability to maintain antimicrobial potential remains questionable and was therefore investigated. We have shown by bottom-up mass spectrometry analysis of protein extracts that both avian beta-defensins AvBD2 and AvBD7 were ubiquitously distributed along the chicken gut. Cathepsin B was found by immunoblotting in jejunum, ileum, caecum, and caecal tonsils, while cathepsins K, L, and S were merely identified in caecal tonsils. Hydrolysis product of AvBD2 and AvBD7 incubated with a panel of proteases was analysed by RP-HPLC, mass spectrometry and antimicrobial assays. AvBD2 and AvBD7 were resistant to serine proteases and to cathepsins D and H. Conversely cysteine cathepsins B, K, L, and S degraded AvBD2 and abolished its antibacterial activity. Only cathepsin K cleaved AvBD7 and released Ile4-AvBD7, a N-terminal truncated natural peptidoform of AvBD7 that displayed antibacterial activity. Besides the 3-stranded antiparallel beta-sheet typical of beta-defensins, structural analysis of AvBD7 by two-dimensional NMR spectroscopy highlighted the restricted accessibility of the C-terminus embedded by the N-terminal region and gave a formal evidence of a salt bridge (Asp9-Arg12) that could account for proteolysis resistance. The differential susceptibility of avian defensins to proteolysis opens intriguing questions about a distinctive role in the mucosal immunity against pathogen invasion.

Human cystatin C: a new biomarker of idiopathic pulmonary fibrosis?

PURPOSE: Human idiopathic pulmonary fibrosis (IPF) is a progressive interstitial lung disorder with a poor prognosis. The identification of a new and specific biomarker in bronchoalveolar lavage fluids (BALFs) may assist in the diagnosis of the disease. EXPERIMENTAL DESIGN: Characterization of cysteine Cats and their endogenous inhibitor, cystatin C, was conducted by immunochemical analysis and measurement of endopeptidase activity of control (n = 11) and IPF (n = 25) BALFs (normalized conditions, 20 µg protein/assay). RESULTS: Cathepsin (Cat) B was detected as proform and mature enzyme for both control and IPF samples, while Cats K, L, and S were found as zymogens with a strengthened staining in IPF BALFs. The overall endopeptidase activity related mainly to Cat B and did not vary significantly between control and IPF samples. Conversely a significant increase of immunoreactive cystatin C was measured in BALFs for each of three IPF grades. CONCLUSIONS AND CLINICAL RELEVANCE: An excessive deposition of extracellular matrix proteins is the hallmark of fibrotic disorders. Cats are potent collagenases and might be essential for lung homeostasis. Taken together, increase of cystatin C in IPF BALFs may reflect abnormal regulation of proteolytic activity of Cats in lung, which in turn can promote the development of fibrosis.

Specific cleavage of the lung surfactant protein A by human cathepsin S may impair its antibacterial properties.

Human cysteine cathepsins (Cats) are implicated in lung injuries and tissue remodeling and have recently emerged as important players in pulmonary inflammations. The proteolytic activities of Cat B, L, K, S and H are dramatically increased in the sputum of patients with cystic fibrosis (CF), suggesting a possible involvement in the CF pathophysiology. We found that pulmonary surfactant protein A (SP-A) that participates to innate host defense is extensively degraded in CF expectorations. Breakdown of SP-A was markedly decreased in CF sputum by E-64 and Mu-Leu-Hph-VSPh, a Cat S inhibitor. Cat S cleaved efficiently and specifically SP-A within critical residues of the solvent-exposed loop of its carbohydrate recognition (C-type lectin) domain that allows binding to pathogens. Cat S decreased aggregation properties of SP-A (self-aggregation, aggregation of phospholipid vesicles and rough LPS). Moreover cleavage of SP-A by Cat S reduced binding to yeast mannan and impaired agglutination of Escherichia coli and Pseudomonas aeruginosa, a foremost detrimental pathogen colonizing the lungs of CF patients. Besides human neutrophil serine proteases and bacterial proteases, we propose that Cat S may participate in the pathophysiology of CF by weakening the antibacterial activity of SP-A. More broadly, present results provide further indication that Cat S, along with Cats B and L, could display immuno-modulatory functions by inactivating key proteins involved in the innate immunity defense.

Intradermal immunization triggers epidermal Langerhans cell mobilization required for CD8 T-cell immune responses.

The potential of the skin immune system for the generation of both powerful humoral and cellular immune responses is now well established. However, the mechanisms responsible for the efficacy of skin antigen-presenting cells (APCs) during intradermal (ID) vaccination still remain to be elucidated. We have previously demonstrated in clinical trials that preferential targeting of Langerhans cells (LCs) by transcutaneous immunization shapes the immune response toward vaccine-specific CD8 T cells. Others have shown that ID inoculation of a vaccine, which targets dermal APCs, mobilizes both the cellular and humoral arms of immunity. Here, we investigated the participation of epidermal LCs in response to ID immunization. When human or mouse skin was injected ID with a particle-based vaccine, we observed significant modifications in the morphology of epidermal LCs and their mobilization to the dermis. We further established that this LC recruitment after ID administration was essential for the induction of antigen-specific CD8 T cells, but was, however, dispensable for the generation of specific CD4 T cells and neutralizing antibodies. Thus, epidermal and dermal APCs shape the outcome of the immune responses to ID vaccination. Their combined potential provides new avenues for the development of vaccination strategies against infectious diseases.

Targeting of HIV-p24 particle-based vaccine into differential skin layers induces distinct arms of the immune responses.

Skin routes of immunization such as subcutaneous (SC), intradermal (ID) and transcutaneous (TC) administration are utilized for vaccination against various pathogens, without understanding their potential impact on the outcome of immune responses. We demonstrated that SC immunization induced HIV-1 p24 specific IgG in absence of antigen-specific CD8 T cells, whereas the ID route induced both cellular and humoral responses. Interestingly, TC application through empty hair follicular ducts, targeting epidermal Langerhans Cells (LCs), induced major CD8 effector cells, in the absence of IgG. However, high levels of mucosal IgA, were localized in the stratified epithelium of the vagina after TC prime. We propose that re-directing the immune responses by targeting differential skin immunization routes, offers enormous potential for innovative vaccination strategies, especially against HIV.

Human cysteine cathepsins are not reliable markers of infection by Pseudomonas aeruginosa in cystic fibrosis.

Cysteine cathepsins have emerged as new players in inflammatory lung disorders. Their activities are dramatically increased in the sputum of cystic fibrosis (CF) patients, suggesting that they are involved in the pathophysiology of CF. We have characterized the cathepsins in CF expectorations and evaluated their use as markers of colonization by Pseudomonas aeruginosa. The concentrations of active cathepsins B, H, K, L and S were the same in P. aeruginosa-positive (19 Ps+) and P. aeruginosa-negative (6 Ps-) samples, unlike those of human neutrophil elastase. Also the cathepsin inhibitory potential and the cathepsins/cathepsin inhibitors imbalance remained unchanged and similar (∼2-fold) in the Ps+ and Ps- groups (p<0.001), which correlated with the breakdown of their circulating cystatin-like inhibitors (kininogens). Procathepsins, which may be activated autocatalytically, are a potential proteolytic reservoir. Immunoblotting and active-site labeling identified the double-chain cathepsin B, the major cathepsin in CF sputum, as the main molecular form in both Ps+ and Ps- samples, despite the possible release of the ∼31 kDa single-chain form from procathepsin B by sputum elastase. Thus, the hydrolytic activity of cysteine cathepsins was not correlated with bacterial colonization, indicating that cathepsins, unlike human neutrophil elastase, are not suitable markers of P. aeruginosa infection.