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Mast cells (MCs) expressing a distinctive protease phenotype (MCTs) selectively expand within the epithelium of human mucosal tissues during type 2 (T2) inflammation. While MCTs are phenotypically distinct from subepithelial MCs (MCTCs), signals driving human MCT differentiation and this subset's contribution to inflammation remain unexplored. Here, we have identified TGF-β as a key driver of the MCT transcriptome in nasal polyps. We found that short-term TGF-β signaling alters MC cell surface receptor expression and partially recapitulated the in vivo MCT transcriptome, while TGF-β signaling during MC differentiation upregulated a larger number of MCT-associated transcripts. TGF-β inhibited the hallmark MCTC proteases chymase and cathepsin G at both the transcript and protein level, allowing selective in vitro differentiation of MCTs for functional study. We identified discrete differences in effector phenotype between in vitro-derived MCTs and MCTCs, with MCTs exhibiting enhanced proinflammatory lipid mediator generation and a distinct cytokine, chemokine, and growth factor production profile in response to both innate and adaptive stimuli, recapitulating functional features of their tissue-associated counterpart MC subsets. Thus, our findings support a role for TGF-β in promoting human MCT differentiation and identified a discrete contribution of this cell type to T2 inflammation.

The genetic causality between cathepsin levels and autoimmune diseases (ADs) bidirectionally was investigated and the associated cancer risk was explored with Mendelian randomization. Mendelian randomization analyses were used to explore causal associations between cathepsin and 14 ADs. The final results came from a meta-analysis of two datasets to get a robust result. Furthermore, the potential carcinogenic effects of reduced cathepsin levels were explored. Sensitivity analyses were used to evaluate the robustness of the results. Based on the Mendelian randomization analysis, it was found that lower levels of specific cathepsins were associated with reduced risk of ADs. Reduced cathepsin E levels were linked to decreased susceptibility to psoriasis and a potential reduction in breast cancer risk. Reduced cathepsins G and L2 showed an inhibitory effect on psoriasis without increasing cancer risk. These results emphasized the genetic causal connection between cathepsin and ADs. Targeting cathepsins may be beneficial in treating ADs, but potential oncogenic effects must be considered to provide a basis for safer therapeutic strategies., Competing Interests: Declarations. Conflict of interest: The authors declare no competing interests., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Molecular dynamics simulations have been remarkably effective for observing and analyzing structures and dynamics of proteins, with longer trajectories being computed every day. Still, often, relevant time scales are not observed. Adequately analyzing the generated trajectories can highlight the interesting areas within a protein such as mutation sites or allosteric hotspots, which might foreshadow dynamics untouched by the simulations. We employ a physics-based protein network and propose that such a network can adequately analyze the protein dynamics. The analysis is conducted on simulations of cathepsin G and neutrophil elastase, which are remarkably similar but with different specificities. However, a single mutation in cathepsin G recovers the specificity of neutrophil elastase. The physics-based network built on the interactions between residues instead of the distances can pinpoint the active triad in the proteins studied. Overall, the network seems to capture the structural behavior better than purely distance-based networks.

Cathepsin G (CTSG) plays an important role in the regulation of immune processes. Accumulated studies show that CTSG is involved in the onset and development of type 1 diabetes mellitus (T1DM). As the genetic background of T1DM varies widely among populations, we aimed to study the relationship between genetic polymorphisms in CTSG and T1DM susceptibility in Chinese populations. A total of 141 patients with T1DM and 200 healthy controls were enrolled in the study. Serum CTSG expression was detected using enzyme-linked immunosorbent assay (ELISA). Genotyping of two selected single nucleotide polymorphisms (SNPs) (rs2236742 and rs2070697) of CTSG was performed using PCR and Sanger sequencing. CTSG expression in patients with T1DM was significantly higher than in the control group. Alleles C and T of CTSG SNP rs2236742 were increased in T1DM. No significant associations were found for the SNP rs2070697. Our results indicate that the CTSG rs2236742 allele (C/T) is associated with T1DM in Chinese children and may serve as a new biomarker for predicting T1DM susceptibility., (© The Author(s) [2024]. Published by Oxford University Press. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Background/aim: Cathepsin G (CTSG) has been identified as an inhibitor of breast, bladder, and colorectal cancers. The G allele of the N125S (A/G, rs45567233) functional polymorphism of the CTSG gene confers increased serum CTSG activity and has been associated with cardiovascular and neurovascular diseases. This study examined the possible correlation between the pathogenesis of basal cell carcinoma (BCC) and the functional polymorphism CTSG N125S., Patients and Methods: A total of 197 DNA samples were examined, comprising 98 BCC patients and 99 control samples of Greek origin. The CTSG N125S polymorphism was molecularly genotyped using PCR amplification, followed by enzyme digestion, and agarose gel electrophoresis of the amplified DNA fragments., Results: There was no statistically significant difference in the genotypic and allelic frequencies between the patient and the control groups., Conclusion: There is no association between the CTSG N125S polymorphism and pathogenesis of BCC., (Copyright © 2024 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Generalized pustular psoriasis (GPP) is an autoinflammatory skin disease whose pathogenesis has not yet been fully elucidated. Alpha-1-antichymotrypsin(ACT) is a protein encoded by the SERPINA3 gene and an inhibitor of cathepsin G. One study of a European sample suggested that the loss of ACT function caused by SERPINA3 mutation is implicated in GPP. However, the role of SERPINA3 in the pathogenesis of GPP in other ethnic populations is unclear. To explore this, seventy children with GPP were performed next-generation sequencing to identify rare variants in the SERPINA3 gene. Bioinformatic analysis and functional tests were used to determine the effects of the variants, and a comprehensive analysis was performed to determine the pathogenicity of the variants and whether they are associated with GPP. One rare deletion and three rare missense variants were identified in the SERPINA3 gene in GPP. The deletion variant c.1246_1247del was found to result in a mutant protein with an extension of 10 amino acids and a C-terminal of 20 amino acids that was completely different from the wild-type. This mutant was found to impede secretion of ACT, thus failing to function as an inhibitor of cathepsin G. Two missense variants were found to reduce the ability of ACT to inhibit cathepsin G enzymatic activity. The association analysis suggested that the deletion variant is associated with GPP. This study identified four rare novel mutations of SERPINA3 and demonstrated that three of these mutations result in loss of function, contributing to the pathogenesis of pediatric-onset GPP in the Asian population., (© 2023. The Author(s), under exclusive licence to The Japan Society of Human Genetics.)

Colorectal cancer (CRC) is the most common gastrointestinal tumor worldwide, which is a severe malignant disease that threatens mankind. Cathepsin G (CTSG) has been reported to be associated with tumorigenesis, whereas its role in CRC is still unclear. This investigation aims to determine the function of CTSG in CRC. Our results indicated that CTSG was inhibited in CRC tissues, and patients with CTSG low expression have poor overall survival. Functional experiments revealed that CTSG overexpression suppressed CRC cell progression in vitro and in vivo, whereas CTSG suppression supports CRC development cells in vitro and in vivo . Mechanistically, CTSG overexpression suppressed Akt/mTOR signaling mechanism and elevated apoptotic-associated markers, and CTSG silencing activated Akt/mTOR signaling mechanisms and inhibited apoptotic-associated markers. Furthermore, the Akt suppression signaling pathway by MK2206 abolishes CTSG-silenced expression-induced cell viability and Bcl2 up-regulation in vitro and in vivo . Altogether, these outcomes demonstrate that CTSG may act as a tumor suppressor gene via Akt/mTOR/Bcl2-mediated anti-apoptotic signaling inactivation, and CTSG represents a potential therapeutic target in CRC., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

The serine proteases neutrophil elastase (NE), proteinase 3 (PR3), cathepsin G (CatG), and neutrophil serine protease 4 (NSP4) are secreted by activated neutrophils as a part of the innate immune response against invading pathogens. However, these serine proteases might be adopted by viruses to mediate viral surface protein priming resulting in host cell entrance and productive infection. Indeed, NE and PR3 hydrolyze the scissile peptide bond within the proteolytically sensitive polybasic sequence of the activation loop of SARS-CoV-2 located at the S1/S2 interface of the Spike (S) protein; an amino acid motif which differs from SARS-CoV-1. The occurrence of novel SARS-CoV-2 variants and substitution of distinct amino acids at the polybasic sequence prompts serious concerns regarding increased transmissibility. We propose that a novel cleavage site by CatG of the Omicron variant and the increased substrate turnover of the Delta variant by furin within the polybasic sequence should be considered for increased transmission of SARS-CoV-2 variants., Competing Interests: The authors declare no conflict of interest.

Inhalation of particulate matter in polluted air causes direct, size-restricted passage in the circulation and pronounced lung inflammation, provoking platelet activation and (non)-fatal cardiovascular complications. To determine potency and mechanism of platelet sensitization via neutrophil enzymes, we performed in vitro aggregation studies in washed human platelets and in murine and human blood, in the presence of elastase, cathepsin G and regular platelet agonists, present in damaged arteries. The impact of both enzymes on in vivo thrombogenicity was studied in the same thrombosis mouse model, previously having demonstrated that neutrophil activation enhances peripheral thrombogenicity. At 0.05 U/mL, cathepsin G activated washed human platelets via PAR1, whereas at 0.35 U/mL, aggregation occurred via PAR4. In Swiss mouse platelet-rich plasma no aggregation occurred by cathepsin G at 0.4 U/mL. In human and murine blood, aggregations by 0.05-0.1 U/mL cathepsin G were similar and not PAR-mediated, but platelet aggregation was inhibited by ADP antagonists, advocating cathepsin G-released ADP in blood as the true agonist of sustained platelet activation. In the mouse thrombosis model, cathepsin G and elastase amplified mild thrombogenicity at blood concentrations that activated platelets in vitro. This study shows that cathepsin G and elastase secreted in the circulation during mild air pollution-induced lung inflammation lyse red blood cell membrane proteins, leading to ADP-leakage into plasma, sensitizing platelets and amplifying their contribution to cardiovascular complications of ambient particle inhalation.

Type 1 diabetes (T1D) is a chronic autoimmune disease accompanied by the immune-mediated destruction of pancreatic β-cells. In this study, we aimed to explore the regulatory effects of vitamin D (VD) supplementation on pancreatic β-cell function by altering the expression of bioinformatically identified cathepsin G (CatG) in T1D mice. A T1D mouse model was established in nonobese diabetic (NOD) mice, and their islets were isolated and purified. Pancreatic mononuclear cells (MNCs) were collected, from which CD4 + T cells were isolated. The levels of interleukin (IL)-2, IL-10, tumor necrosis factor-α (TNF-α), and interferon-γ (IFN-γ) in the supernatant of mouse pancreatic tissue homogenate were assessed using ELISA. Immunohistochemistry and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labelin (TUNEL) staining were conducted to evaluate the effects of VD supplementation on pancreatic tissues of T1D mice. The pancreatic β-cell line MIN6 was used for in vitro substantiation of findings in vivo. VD supplementation reduced glucose levels and improved glucose tolerance in T1D mice. Furthermore, VD supplementation improved pancreatic β-cell function and suppressed immunological and inflammatory reactions in the T1D mice. We documented overexpression of CatG in diabetes tissue samples, and then showed that VD supplementation normalized the islet immune microenvironment through downregulating CatG expression in T1D mice. Experiments in vitro subsequently demonstrated that VD supplementation impeded CD4 + T activation by downregulating CatG expression and thereby enhanced pancreatic β-cell function. Results of the present study elucidated that VD supplementation can downregulate the expression of CatG and inhibit CD4 + T cell activation, thereby improving β-cell function in T1D. NEW & NOTEWORTHY We report that vitamin D (VD) supplementation downregulates CatG expression and inhibits CD4 + T cell activation, thereby improving β-cell function in type 1 diabetes (T1D). This study deepens our understanding of the pathogenesis of T1D and clarifies molecular events underlying the alleviatory effect of VD for immunotherapy against T1D.

Recently, we reported a case of an infant with neonatal severe under-mineralizing skeletal dysplasia caused by mutations within both alleles of the TRPV6 gene. One mutation results in an in frame stop codon (R 510 stop) that leads to a truncated, nonfunctional TRPV6 channel, and the second in a point mutation (G 660 R) that, surprisingly, does not affect the Ca 2+ permeability of TRPV6. We mimicked the subunit composition of the unaffected heterozygous parent and child by coexpressing the TRPV6 G 660 R and R 510 stop mutants and combinations with wild type TRPV6. We show that both the G 660 R and R 510 stop mutant subunits are expressed and result in decreased calcium uptake, which is the result of the reduced abundancy of functional TRPV6 channels within the plasma membrane. We compared the proteomic profiles of a healthy placenta with that of the diseased infant and detected, exclusively in the latter two proteases, HTRA1 and cathepsin G. Our results implicate that the combination of the two mutant TRPV6 subunits, which are expressed in the placenta of the diseased child, is responsible for the decreased calcium uptake, which could explain the skeletal dysplasia. In addition, placental calcium deficiency also appears to be associated with an increase in the expression of proteases.

Background: This study investigated the expression and localization of cathepsins B, D, and G in relationship to the embryonic stem cell (ESC)-like population we have previously identified in microcystic lymphatic malformation (mLM). Methods and Results: Immunohistochemical staining demonstrated expression of cathepsins B, D, and G in cervicofacial mLM tissue samples from 11 patients. Immunofluorescence staining of two representative mLM samples showed localization of cathepsins B and D to the OCT4+ and the c-MYC+ cells on the endothelium of lesional vessels and the stroma, while cathepsin G was localized to the OCT4+/tryptase+ cells within the stroma. Transcript expression of cathepsins B, D, and G was confirmed using reverse transcription quantitative polymerase chain reaction (RT-qPCR; n  = 5). Western blotting ( n  = 3) performed on the mLM tissue samples revealed protein expression of cathepsins B and D, which were demonstrated to be enzymatically active using enzymatic activity assays. Conclusion: This study demonstrated expression of cathepsins B and D by the ESC-like cells on the endothelium of lesional vessels and the stroma, while cathepsin G was localized to the OCT4+ phenotypic mast cells within the stroma of mLM.

Chromatin undergoes extensive reprogramming during immune cell differentiation. Here we report the repression of controlled histone H3 amino terminus proteolytic cleavage (H3ΔN) during monocyte-to-macrophage development. This abundant histone mark in human peripheral blood monocytes is catalyzed by neutrophil serine proteases (NSPs) cathepsin G, neutrophil elastase and proteinase 3. NSPs are repressed as monocytes mature into macrophages. Integrative epigenomic analysis reveals widespread H3ΔN distribution across the genome in a monocytic cell line and primary monocytes, which becomes largely undetectable in fully differentiated macrophages. H3ΔN is enriched at permissive chromatin and actively transcribed genes. Simultaneous NSP depletion in monocytic cells results in H3ΔN loss and further increase in chromatin accessibility, which likely primes the chromatin for gene expression reprogramming. Importantly, H3ΔN is reduced in monocytes from patients with systemic juvenile idiopathic arthritis, an autoinflammatory disease with prominent macrophage involvement. Overall, we uncover an epigenetic mechanism that primes the chromatin to facilitate macrophage development.

Dermatomyositis (DM) is a multifactorial chronic autoimmune disorder with characteristic skin and muscle pathological changes and involvement of other organ systems. Cathepsin G (CTSG) contributes to the risk of developing DM, which is likely to be associated with inflammatory cytokines. Differential DNA methylation on CTSG has been determined to be implicated in DM in vivo. However, the underlying mechanism of this epigenetic regulation on CTST in DM is poorly explored. In this study, we investigated DNA methylation signature on CTSG at single-nucleotide resolution in quadriceps femoris of six DM patients and paracancerous muscles of three patients with rhabdomyosarcoma on inner thigh using pyrosequencing and observed that the overall DNA methylation level of CTSG was increased in DM compared with control, in which CpG loci at third and fourth exons but not promoter contributed to the significant hypermethylation. Furthermore, we observed that transcription and DNA methylation of CTSG were both declined in DNMT3a knockdown compared with DNMT1 and DNMT3b knockdown in human skeletal muscle SJCRH30 and A-204 cell lines exposed to tumor necrosis factor-α. Furthermore, Bortezomib (NF-κB inhibitor) and Brevilin A (JAK/STAT inhibitor) were employed to treat SJCRH30 and A-204 cells, respectively, and we observed that CTSG was hypomethylated and silenced after Bortezomib treatment compared with untreatment and Brevilin A. Finally, chromatin immunoprecipitation-quantitative polymerase chain reaction indicated that DNMT3a could bind to the coding regions of CTSG and the interaction was dependent on NF-κB activity. Taken together, our results determined a novel regulatory mechanism of DNA methylation on CTSG in DM., (© 2020 International Federation for Cell Biology.)

Background and Purpose: Inflammation has emerged as a key component of the pathophysiology of intracranial aneurysms. Mast cells have been detected in human intracranial aneurysm tissues, and their presence was associated with intramural microhemorrhage and wall degeneration. We hypothesized that mast cells play a critical role in the development of aneurysmal rupture, and that mast cells can be used as a therapeutic target for the prevention of aneurysm rupture., Methods: Intracranial aneurysms were induced in adult mice using a combination of induced systemic hypertension and a single injection of elastase into the cerebrospinal fluid. Aneurysm formation and rupture were assessed over 3 weeks. Roles of mast cells were assessed using a mast cell stabilizer (cromolyn), a mast cell activator (C48/80), and mice that are genetically lacking mature mast cells (Kit W-sh/W-sh mice)., Results: Pharmacological stabilization of mast cells with cromolyn markedly decreased the rupture rate of aneurysms (80% versus 19%, n=10 versus n =16) without affecting the aneurysm formation. The activation of mast cells with C48/80 significantly increased the rupture rate of aneurysms (25% versus 100%, n=4 versus n=5) without affecting the overall rate of aneurysm formation. Furthermore, the genetic deficiency of mast cells significantly prevented aneurysm rupture (80% versus 25%, n=10 versus n=8, wild-type versus Kit W-sh/W-sh mice)., Conclusions: These results suggest that mast cells play a key role in promoting aneurysm rupture but not formation. Stabilizers of mast cells may have a potential therapeutic value in preventing intracranial aneurysm rupture in patients.

Sepsis is a life-threatening disorder and leads to organ dysfunction and death. Therefore, searching for more alternative biomarkers is of great significance for sepsis assessment and surveillance. In our study, the gene expression profiles of 163 samples from healthy controls and septic patients were analyzed and 8 gene co-expression modules were identified by constructing weighted gene co-expression network. The blue and yellow modules showed close correlations with the phenotypic trait "days postsepsis." Besides, differentially expressed genes (DEGs) over time in septic patients were screened using Short Time-series Expression Miner (STEM) program. The intersection of genes in the blue and yellow modules and DEGs, which were significantly enriched in "HTLV-1 infection" pathway, was analyzed with protein-protein interaction network. The logistic regression model based on these eight mRNAs was constructed to determine the type of the sample reliably. Eight vital genes CECR1 , ANXA2 , ELANE , CTSG , AZU1 , PRTN3 , LYZ , and DEFA4 presented high scores and may be associated with sepsis, which provided candidate biomarkers for sepsis.

To identify an immune-related prognostic signature based on long non-coding RNAs (lncRNAs) and find immunotherapeutic targets for bladder urothelial carcinoma, we downloaded RNA-sequencing data from The Cancer Genome Atlas (TCGA) dataset. Functional enrichment analysis demonstrated bladder urothelial carcinoma was related to immune-related functions. We obtained 332 immune-related genes and 262 lncRNAs targeting immune-related genes. We constructed a signature based on eight lncRNAs in training cohort. Patients were classified as high-risk and low-risk according to signature risk score. High-risk patients had poor overall survival compared with low-risk patients ( P < 0.001). Multivariate Cox regression suggested the signature was an independent prognostic indicator. The findings were further validated in testing, entire TCGA and external validation cohorts. Gene set enrichment analysis indicated significant enrichment of immune-related phenotype in high-risk group. Immunohistochemistry and online analyses validated the functions of 4 key immune-related genes (LIG1, TBX1, CTSG and CXCL12) in bladder urothelial carcinoma. Nomogram proved to be a good classifier for muscle-invasive bladder cancer through combining the signature. In conclusion, our immune-related prognostic signature and nomogram provided prognostic indicators and potential immunotherapeutic targets for muscle-invasive bladder cancer.

Polymorphonuclear granulocytes (PMNs) are indispensable for controlling life-threatening fungal infections. In addition to various effector mechanisms, PMNs also produce extracellular vesicles (EVs). Their contribution to antifungal defense has remained unexplored. We reveal that the clinically important human-pathogenic fungus Aspergillus fumigatus triggers PMNs to release a distinct set of antifungal EVs (afEVs). Proteome analyses indicated that afEVs are enriched in antimicrobial proteins. The cargo and the release kinetics of EVs are modulated by the fungal strain confronted. Tracking of afEVs indicated that they associated with fungal cells and even entered fungal hyphae, resulting in alterations in the morphology of the fungal cell wall and dose-dependent antifungal effects. To assess as a proof of concept whether the antimicrobial proteins found in afEVs might contribute to growth inhibition of hyphae when present in the fungal cytoplasm, two human proteins enriched in afEVs, cathepsin G and azurocidin, were heterologously expressed in fungal hyphae. This led to reduced fungal growth relative to that of a control strain producing the human retinol binding protein 7. In conclusion, extracellular vesicles produced by neutrophils in response to A. fumigatus infection are able to associate with the fungus, limit growth, and elicit cell damage by delivering antifungal cargo. This finding offers an intriguing, previously overlooked mechanism of antifungal defense against A. fumigatus IMPORTANCE Invasive fungal infections caused by the mold Aspergillus fumigatus are a growing concern in the clinic due to the increasing use of immunosuppressive therapies and increasing antifungal drug resistance. These infections result in high rates of mortality, as treatment and diagnostic options remain limited. In healthy individuals, neutrophilic granulocytes are critical for elimination of A. fumigatus from the host; however, the exact extracellular mechanism of neutrophil-mediated antifungal activity remains unresolved. Here, we present a mode of antifungal defense employed by human neutrophils against A. fumigatus not previously described. We found that extracellular vesicles produced by neutrophils in response to A. fumigatus infection are able to associate with the fungus, limit growth, and elicit cell damage by delivering antifungal cargo. In the end, antifungal extracellular vesicle biology provides a significant step forward in our understanding of A. fumigatus host pathogenesis and opens up novel diagnostic and therapeutic possibilities., (Copyright © 2020 Shopova et al.)

Acute pancreatitis is characterized by an early intracellular protease activation and invasion of leukocytes into the pancreas. Cathepsins constitute a large group of lysosomal enzymes, that have been shown to modulate trypsinogen activation and neutrophil infiltration. Cathepsin G (CTSG) is a neutrophil serine protease of the chymotrypsin C family known to degrade extracellular matrix components and to have regulatory functions in inflammatory disorders. The aim of this study was to investigate the role of CTSG in pancreatitis. Isolated acinar cells were exposed to recombinant CTSG and supramaximal cholezystokinin stimulation. In CTSG -/- mice and corresponding controls acute experimental pancreatitis was induced by serial caerulein injections. Severity was assessed by histology, serum enzyme levels and zymogen activation. Neutrophil infiltration was quantified by chloro-acetate ersterase staining and myeloperoxidase measurement. CTSG was expessed in inflammatory cells but not in pancreatic acinar cells. CTSG had no effect on intra-acinar-cell trypsinogen activation. In CTSG -/- mice a transient decrease of neutrophil infiltration into the pancreas and lungs was found during acute pancreatitis while the disease severity remained largely unchanged. CTSG is involved in pancreatic neutrophil infiltration during pancreatitis, albeit to a lesser degree than the related neutrophil (PMN) elastase. Its absence therefore leaves pancreatitis severity essentially unaffected.

Background: Osteomyelitis is a bone infection, most often caused by Staphylococcus aureus, in which neutrophils play a key role. Cathepsin G (CTSG) is a bactericidal serine protease stored in the neutrophil azurophilic granules. CTSG regulates inflammation, activating matrix metalloproteinases (MMPs), and coagulation. Lactoferrin (LF), a neutrophil glycoprotein, increases CTSG catalytic activity and induces inflammation. The aim of this study was to analyze a potential association between a CTSG gene polymorphism (Asn125Ser or N125S, rs45567233), that modifies CTSG activity, and could affect susceptibility to, or outcome of, bacterial osteomyelitis., Methods: CTSG N125S polymorphism was genotyped in 329 osteomyelitis patients and 415 controls), Blood coagulation parameters, serum CTSG activity, LF, MMP-1, MMP-13, and soluble receptor activator for nuclear factor κ B ligand (sRANKL) levels were assessed in carriers of the different CTSG genotypes., Results: CTSG N125S (AG) genotype was significantly more frequent among osteomyelitis patients than controls (15.5% vs. 9.4%, p = 0.014). CTSG N125S variant G allele (AG +GG) was also more frequent among osteomyelitis patients (8.1% vs. 4.7%, p = 0.01). Serum CTSG activity and LF levels were significantly higher in osteomyelitis patients carrying the G allele compared to those with the AA genotype, (p<0.04). Serum MMP-1 was lower in the G allele carriers (p = 0.01). There was no association between these genotypes and clinical characteristics of osteomyelitis, or coagulation parameters, MMP-13, and sRANKL serum levels., Conclusions: Differences in the CTSG gene might enhance osteomyelitis susceptibility by increasing CTSG activity and LF levels., Competing Interests: The authors have declared that no competing interests exist.

During infection, increasing pathogen load stimulates both protective and harmful aspects of the host response. The dynamics of this interaction are hard to quantify in humans, but doing so could improve understanding of the mechanisms of disease and protection. We sought to model the contributions of the parasite multiplication rate and host response to observed parasite load in individual subjects infected with Plasmodium falciparum malaria, using only data obtained at the time of clinical presentation, and then to identify their mechanistic correlates. We predicted higher parasite multiplication rates and lower host responsiveness in cases of severe malaria, with severe anaemia being more insidious than cerebral malaria. We predicted that parasite-growth inhibition was associated with platelet consumption, lower expression of CXCL10 and type 1 interferon-associated genes, but increased cathepsin G and matrix metallopeptidase 9 expression. We found that cathepsin G and matrix metallopeptidase 9 directly inhibit parasite invasion into erythrocytes. The parasite multiplication rate was associated with host iron availability and higher complement factor H levels, lower expression of gametocyte-associated genes but higher expression of translation-associated genes in the parasite. Our findings demonstrate the potential of using explicit modelling of pathogen load dynamics to deepen understanding of host-pathogen interactions and identify mechanistic correlates of protection.

Although studies have provided significant evidence about the role of RAS in mediating cancer risk in type 2 diabetes mellitus (DM), conclusions about the central molecular mechanisms underlying this disease remain to be reached, because this type of information requires an integrative multi-omics approach. In the current study, meta-analysis was performed on type 2 diabetes and breast, bladder, liver, pancreas, colon and rectum cancer-associated transcriptome data, and reporter biomolecules were identified at RNA, protein, and metabolite levels using the integration of gene expression profiles with genome-scale biomolecular networks in diabetes samples. This approach revealed that RAS biomarkers could be associated with cancer initiation and progression, which include metabolites (particularly, aminoacyl-tRNA biosynthesis and ABC transporters) as novel biomarker candidates and potential therapeutic targets. We detected downregulation and upregulation of differentially expressed genes (DEGs) in blood, pancreatic islets, liver and skeletal muscle from normal and diabetic patients. DEGs were combined with 211 renin-angiotensin-system related genes. Upregulated genes were enriched using Pathway analysis of cancer in pancreatic islets, blood and skeletal muscle samples. It seems that the changes in mRNA are contributing to the phenotypic changes in carcinogenesis, or that they are as a result of the phenotypic changes associated with the malignant transformation. Our analyses showed that Ctsg and Ednrb are downregulated in cancer samples. However, by immunohistochemistry experiments we observed that EDNRB protein showed increased expression in tumor samples. It is true that alterations in mRNA expression do not always reflect alterations in protein expression, since post-translational changes can occur in proteins. In this study, we report valuable data for further experimental and clinical analysis, because the proposed biomolecules have significant potential as systems biomarkers for screening or for therapeutic purposes in type 2 diabetes and cancer-associated pathways., (Copyright © 2019 Elsevier B.V. All rights reserved.)

While proteases are essential in gastrointestinal physiology, accumulating evidence indicates that dysregulated proteolysis plays a pivotal role in the pathophysiology of inflammatory bowel disease (IBD). Nonetheless, the identity of overactive proteases released by human colonic mucosa remains largely unknown. Studies of protease abundance have primarily investigated expression profiles, not taking into account their enzymatic activity. Herein we have used serine protease-targeted activity-based probes (ABPs) coupled with mass spectral analysis to identify active forms of proteases secreted by the colonic mucosa of healthy controls and IBD patients. Profiling of (Pro-Lys)-ABP bound proteases revealed that most of hyperactive proteases from IBD secretome are clustered at 28-kDa. We identified seven active proteases: the serine proteases cathepsin G, plasma kallikrein, plasmin, tryptase, chymotrypsin-like elastase 3 A, and thrombin and the aminopeptidase B. Only cathepsin G and thrombin were overactive in supernatants from IBD patient tissues compared to healthy controls. Gene expression analysis highlighted the transcription of genes encoding these proteases into intestinal mucosae. The functional ABP-targeted proteomic approach that we have used to identify active proteases in human colonic samples bears directly on the understanding of the role these enzymes may play in the pathophysiology of IBD.

Background: Mast cells are key effector cells in allergic reactions. When activated to degranulate, they release a plethora of bioactive compounds from their secretory granules, including mast cell-restricted proteases such as tryptase. In a previous study, we showed that tryptase, in addition to its intragranular location, can be found within the nuclei of mast cells where it truncates core histones at their N-terminal ends., Objective: Considering that the N-terminal portions of the core histones constitute sites for posttranslational modifications of major epigenetic impact, we evaluated whether histone truncation by tryptase could have an impact on epigenetic events in mast cells., Methods: Mast cells were cultured from wild-type and tryptase null mice, followed by an assessment of their profile of epigenetic histone modifications and their phenotypic characteristics., Results: We show that tryptase truncates nucleosomal histone 3 and histone 2B (H2B) and that its absence results in accumulation of the epigenetic mark, lysine 5-acetylated H2B. Intriguingly, the accumulation of lysine 5-acetylated H2B was cell age-dependent and was associated with a profound upregulation of markers of non-mast cell lineages, loss of proliferative control, chromatin remodeling as well as extensive morphological alterations., Conclusions: These findings introduce tryptase-catalyzed histone clipping as a novel epigenetic regulatory mechanism, which in the mast cell context may be crucial for maintaining cellular identity., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

The efficacy of B cell depletion therapy in multiple sclerosis indicates their central pathogenic role in disease pathogenesis. The B lymphotropic EBV is a major risk factor in multiple sclerosis, via as yet unclear mechanisms. We reported in a nonhuman primate experimental autoimmune encephalomyelitis model that an EBV-related lymphocryptovirus enables B cells to protect a proteolysis-sensitive immunodominant myelin oligodendrocyte glycoprotein (MOG) epitope (residues 40-48) against destructive processing. This facilitates its cross-presentation to autoaggressive cytotoxic MHC-E-restricted CD8 + CD56 + T cells. The present study extends these observations to intact human B cells and identifies a key role of autophagy. EBV infection upregulated APC-related markers on B cells and activated the cross-presentation machinery. Although human MOG protein was degraded less in EBV-infected than in uninfected B cells, induction of cathepsin G activity by EBV led to total degradation of the immunodominant peptides MOG 35-55 and MOG 1-20 Inhibition of cathepsin G or citrullination of the arginine residue within an LC3-interacting region motif of immunodominant MOG peptides abrogated their degradation. Internalized MOG colocalized with autophagosomes, which can protect from destructive processing. In conclusion, EBV infection switches MOG processing in B cells from destructive to productive and facilitates cross-presentation of disease-relevant epitopes to CD8 + T cells., (Copyright © 2017 by The American Association of Immunologists, Inc.)

Estrogen, a natural immunomodulator, regulates the development and function of diverse immune cell types. There is now renewed attention on neutrophils and neutrophil serine proteases (NSPs) such as neutrophil elastase (NE), proteinase 3 (PR3), and cathepsin G (CG) in inflammation and autoimmunity. In this study, we found that although estrogen treatment significantly reduced total splenocytes number, it markedly increased the splenic neutrophil absolute numbers in estrogen-treated C57BL/6 (B6) mice when compared to placebo controls. Concomitantly, the levels of NSPs and myeloperoxidase (MPO) were highly upregulated in the splenocytes from estrogen-treated mice. Despite the critical role of NSPs in the regulation of non-infectious inflammation, by employing NE-/-/PR3-/-/CG-/- triple knock out mice, we demonstrated that the absence of NSPs affected neither estrogen's ability to increase splenic neutrophils nor the induction of inflammatory mediators (IFNγ, IL-1β, IL-6, TNFα, MCP-1, and NO) from ex vivo activated splenocytes. Depletion of neutrophils in vitro in splenocytes with anti-Ly6G antibody also had no obvious effect on NSP expression or LPS-induced IFNγ and MCP-1. These data suggest that estrogen augments NSPs, which appears to be independent of enhancing ex vivo inflammatory responses. Since estrogen has been implicated in regulating several experimental autoimmune diseases, we extended our observations in estrogen-treated B6 mice to spontaneous autoimmune-prone female MRL-lpr, B6-lpr and NZB/WF1 mice. There was a remarkable commonality with regards to the increase of neutrophils and concomitant increase of NSPs and MPO in the splenic cells of different strains of autoimmune-prone mice and estrogen-treated B6 mice. Collectively, since NSPs and neutrophils are involved in diverse pro-inflammatory activities, these data suggest a potential pathologic implication of increased neutrophils and NSPs that merits further investigation., Competing Interests: The authors have declared that no competing interests exist.

Here, we evaluated whether the overexpression of transcriptionally inactive ΔNp73 cooperates with PML/RARA fusion protein in the induction of an APL-leukemic phenotype, as well as its role in vitro in proliferation, myeloid differentiation, and drug-induced apoptosis. Using lentiviral gene transfer, we showed in vitro that ΔNp73 overexpression resulted in increased proliferation in murine bone marrow (BM) cells from hCG-PML/RARA transgenic mice and their wild-type (WT) counterpart, with no accumulation of cells at G2/M or S phases; instead, ΔNp73-expressing cells had a lower rate of induced apoptosis. Next, we evaluated the effect of ΔNp73 on stem-cell self-renewal and myeloid differentiation. Primary BM cells lentivirally infected with human ΔNp73 were not immortalized in culture and did not present significant changes in the percentage of CD11b. Finally, we assessed the impact of ΔNp73 on leukemogenesis or its possible cooperation with PML/RARA fusion protein in the induction of an APL-leukemic phenotype. After 120 days of follow-up, all transplanted mice were clinically healthy and, no evidence of leukemia/myelodysplasia was apparent. Taken together, our data suggest that ΔNp73 had no leukemic transformation capacity by itself and apparently did not cooperate with the PML/RARA fusion protein to induce a leukemic phenotype in a murine BM transplantation model. In addition, the forced expression of ΔNp73 in murine BM progenitors did not alter the ATRA-induced differentiation rate in vitro or induce aberrant cell proliferation, but exerted an important role in cell survival, providing resistance to drug-induced apoptosis.

Major histocompatibility complex (MHC) class I molecules present antigenic peptides to cytotoxic T cells. During an adaptive immune response, MHC molecules are regulated by several mechanisms including lipopolysaccharide (LPS) and interferon gamma (IFN-g). However, it is unclear whether the serine protease cathepsin G (CatG), which is generally secreted by neutrophils at the site of inflammation, might regulate MHC I molecules. We identified CatG, and to a higher extend CatG and lactoferrin (LF), as an exogenous regulator of cell surface MHC I expression of immune cells and glioblastoma stem cells. In addition, levels of MHC I molecules are reduced on dendritic cells from CatG deficient mice compared to their wild type counterparts. Furthermore, cell surface CatG on immune cells, including T cells, B cells, and NK cells triggers MHC I on THP-1 monocytes suggesting a novel mechanism for CatG to facilitate intercellular communication between infiltrating cells and the respective target cell. Subsequently, our findings highlight the pivotal role of CatG as a checkpoint protease which might force target cells to display their intracellular MHC I:antigen repertoire.

Background: Therapeutic targeting of arterial leukocyte recruitment in the context of atherosclerosis has been disappointing in clinical studies. Reasons for such failures include the lack of knowledge of arterial-specific recruitment patterns. Here we establish the importance of the cathepsin G (CatG) in the context of arterial myeloid cell recruitment., Methods: Intravital microscopy of the carotid artery, the jugular vein, and cremasteric arterioles and venules in Apoe -/- and CatG-deficient mice (Apoe -/- Ctsg -/- ) was used to study site-specific myeloid cell behavior after high-fat diet feeding or tumor necrosis factor stimulation. Atherosclerosis development was assessed in aortic root sections after 4 weeks of high-fat diet, whereas lung inflammation was assessed after inhalation of lipopolysaccharide. Endothelial deposition of CatG and CCL5 was quantified in whole-mount preparations using 2-photon and confocal microscopy., Results: Our observations elucidated a crucial role for CatG during arterial leukocyte adhesion, an effect not found during venular adhesion. Consequently, CatG deficiency attenuates atherosclerosis but not acute lung inflammation. Mechanistically, CatG is immobilized on arterial endothelium where it activates leukocytes to firmly adhere engaging integrin clustering, a process of crucial importance to achieve effective adherence under high-shear flow. Therapeutic neutralization of CatG specifically abrogated arterial leukocyte adhesion without affecting myeloid cell adhesion in the microcirculation. Repetitive application of CatG-neutralizing antibodies permitted inhibition of atherogenesis in mice., Conclusions: Taken together, these findings present evidence of an arterial-specific recruitment pattern centered on CatG-instructed adhesion strengthening. The inhibition of this process could provide a novel strategy for treatment of arterial inflammation with limited side effects., (© 2016 American Heart Association, Inc.)

Objective/background: The aim was to investigate the expression of genes associated with carotid plaque instability and their protein products at a local and systemic level., Methods: Carotid plaques from 24 patients undergoing carotid endarterectomy (CEA) were classified as stable or unstable using clinical, histological, ultrasound, and transcranial Doppler criteria, and compared using whole genome microarray chips. Initial results of differentially expressed genes were validated by quantitative reverse transcriptase polymerase chain reaction in an independent group of 96 patients undergoing CEA. The protein product of genes significantly differentially expressed between patients with stable and unstable plaques were analysed by plaque immunohistochemistry and serum protein quantification by enzyme-linked immunosorbent assay on a further independent cohort., Results: Expression of chemokine (c-c-motif) ligand 19 (CCL19) was significantly upregulated in plaques from patients with clinically unstable disease (p < .001). Cathepsin G expression was upregulated in histologically unstable plaques (p = .04). Serum concentration of CCL19 was significantly higher in patients with clinically unstable plaques (p = .02). Immunohistochemical staining for CCL19 demonstrated positive staining in histologically and clinically unstable plaques (p = .03). CCL19 also co-localised with CD3 + T-cell lymphocytes in the core region, around where CCL19 was expressed., Conclusions: CCL19 is significantly overexpressed in patients with unstable carotid atherosclerotic plaques and may be a possible novel biomarker for identifying high-risk patients in whom more urgent intervention may be indicated., (Copyright © 2016 European Society for Vascular Surgery. Published by Elsevier Ltd. All rights reserved.)

Neutrophil serine proteases, such as cathepsin G (CG) and neutrophil elastase (NE), have been implicated in the protective response against infections, including experimental mycobacterial infections. The goal of this study was to explore the role of CG in immunocompetent mice challenged aerogenically with Mycobacterium tuberculosis. We used genetically CG- or CG/NE-deficient mice to define the importance of these neutrophil serine proteases for antibacterial protection, granulomatous response, and survival. In addition, we explored the effect of intratracheally delivered liposomally encapsulated CG/NE as a therapeutic approach early during M. tuberculosis infection. Our data show that the presence of CG or CG/NE prolongs survival in M. tuberculosis-infected mice. However, CG is not directly involved in antibacterial defenses, and exogenous intratracheal administration of CG combined with NE does not reduce bacterial loads in the lungs of M. tuberculosis-infected mice., (Copyright © 2015 by The American Association of Immunologists, Inc.)

Background: Proteases have been shown to modulate pain signaling in the spinal cord and may contribute to the development of chronic postsurgical pain. By using peripheral inflammation in rats as a chronic pain model, the authors identified the deregulation of proteases and their inhibitors as a hallmark of chronic pain development using a genome-wide screening approach., Methods: A microarray analysis was performed and identified spinal cathepsin G (CTSG) as the most up-regulated gene in rats with persistent hyperalgesia after intraplantar injection of complete Freund's adjuvant (n = 4). Further experiments were performed to elucidate the mechanisms of CTSG-induced hyperalgesia by intrathecally applying specific CTSG inhibitor (n = 10). The authors also evaluated the association between CTSG gene polymorphisms and the risk of chronic postsurgical pain in 1,152 surgical patients., Results: CTSG blockade reduced heat hyperalgesia, accompanied by a reduction in neutrophil infiltration and interleukin 1β levels in the dorsal horns. In the gene association study, 246 patients (21.4%) reported chronic postsurgical pain at 12-month follow-up. Patients with AA genotypes at polymorphisms rs2070697 (AA-15.3%, GA-24.1%, and GG-22.3%) or rs2236742 (AA-6.4%, GA-20.4%, and GG-22.6%) in the CTSG gene had lower risk for chronic postsurgical pain compared with wild-types. The adjusted odds ratios were 0.67 (95% CI, 0.26 to 0.99) and 0.34 (95% CI, 0.21 to 0.98), respectively., Conclusions: This study demonstrated that CTSG is a pronociceptive mediator in both animal model and human study. CTSG represents a new target for pain control and a potential marker to predict patients who are prone to develop chronic pain after surgery.

The α-defensins, human neutrophil peptides (HNPs) are the predominant antimicrobial peptides of neutrophil granules. They are synthesized in promyelocytes and myelocytes as proHNPs, but only processed in promyelocytes and stored as mature HNPs in azurophil granules. Despite decades of search, the mechanisms underlying the posttranslational processing of neutrophil defensins remain unidentified. Thus, neither the enzyme that processes proHNPs nor the localization of processing has been identified. It has been hypothesized that proHNPs are processed by the serine proteases highly expressed in promyelocytes: Neutrophil elastase (NE), cathepsin G (CG), and proteinase 3 (PR3), all of which are able to process recombinant proHNP into HNP in vitro. We investigated whether serine proteases are in fact responsible for processing of proHNP in human bone marrow cells and in human and murine myeloid cell lines. Subcellular fractionation of the human promyelocytic cell line PLB-985 demonstrated proHNP processing to commence in fractions containing endoplasmic reticulum. Processing of 35S-proHNP was insensitive to serine protease inhibitors. Simultaneous knockdown of NE, CG, and PR3 did not decrease proHNP processing in primary human bone marrow cells. Furthermore, introduction of NE, CG, and PR3 into murine promyelocytic cells did not enhance the proHNP processing capability. Finally, two patients suffering from Papillon-Lefèvre syndrome, who lack active neutrophil serine proteases, demonstrated normal levels of fully processed HNP in peripheral neutrophils. Contradicting earlier assumptions, our study found serine proteases dispensable for processing of proHNPs in vivo. This calls for study of other protease classes in the search for the proHNP processing protease(s).

Angiotensin II favors the development of atherosclerosis. Our goal was to determine if foam cell formation increases angiotensin II generation by the endogenous renin-angiotensin system (RAS) and if endogenously produced angiotensin II promotes lipid accumulation in macrophages. Differentiated THP-1 cells were treated with acetylated low-density lipoproteins (ac-LDL), native LDL (n-LDL), or no LDL. Expression of RAS genes was assessed and angiotensin I/II levels were quantified in media and cell lysate. Ac-LDL increased angiotensin I/II levels and the angiotensin II/I ratio in cells and media after foam cell formation. Renin mRNA or activity did not change, but renin blockade completely inhibited the increase in angiotensin II. Angiotensinogen mRNA but not protein level was increased. Angiotensin-converting enzyme (ACE) and cathepsin G mRNA and activities were enhanced by ac-LDL. Inhibition of renin, ACE, or the angiotensin II receptor 1 (AT1-receptor) largely abolished cholesteryl ester formation in cells exposed to ac-LDL and decreased scavenger receptor A (SR-A) and acyl-coenzyme A:cholesterol acyltransferase 1 (ACAT-1) protein levels. Inhibition of renin or the AT1-receptor in cells treated with oxidized LDL also decreased SR-A and ACAT-1 protein and foam cell formation. ac-LDL also increased angiotensin II by human peripheral blood monocyte-derived macrophages, whereas blockade of renin decreased cholesterol ester formation in these macrophages. These findings indicate that, during foam cell formation, angiotensin II generation by the endogenous RAS is stimulated and that endogenously generated angiotensin II is crucial for cholesterol ester accumulation in macrophages exposed to modified LDL.

Cathepsin G is a serine protease with a broad range of catalytic activities, including production of angiotensin II, degradation of extracellular matrix and cell-cell junctions, modulation of chemotactic responses, and induction of apoptosis. Cathepsin G mRNA expression is increased in human coronary atheroma vs. the normal vessel. To assess whether cathepsin G modulates atherosclerosis, cathepsin G knockout (Cstg(-/-)) mice were bred with apolipoprotein E knockout (Apoe(-/-)) mice to obtain Ctsg(+/-)Apoe(-/-) and Ctsg(+/+)Apoe(-/-) mice. Heterozygous cathepsin G deficiency led to a 70% decrease in cathepsin G activity in bone marrow cells, but this reduced activity did not impair generation of angiotensin II in bone marrow-derived macrophages (BMDM). Atherosclerotic lesions were compared in male Cstg(+/-)Apoe(-/-) and Cstg(+/+)Apoe(-/-) mice after 8 wk on a high-fat diet. Plasma cholesterol levels and cholesterol distribution within serum lipoprotein fractions did not differ between genotypes nor did the atherosclerotic lesion areas in either the aortic root or aortic arch. Cstg(+/-)Apoe(-/-) mice, however, showed a lower percentage of complex lesions within the aortic root and a smaller number of apoptotic cells compared with Cstg(+/+)Apoe(-/-) littermates. Furthermore, apoptotic Cstg(-/-) BMDM were more efficiently engulfed by phagocytic BMDM than were apoptotic Ctsg(+/+) BMDM. Thus cathepsin G activity may impair efferocytosis, which could lead to an accumulation of lesion-associated apoptotic cells and the accelerated progression of early atherosclerotic lesions to more complex lesions in Apoe(-/-) mice.

Acute promyelocytic leukemia (APL) is initiated by the PML-RARA (PR) fusion oncogene and has a characteristic expression profile that includes high levels of the Notch ligand Jagged-1 (JAG1). In this study, we used a series of bioinformatic, in vitro, and in vivo assays to assess the role of Notch signaling in human APL samples, and in a PML-RARA knock-in mouse model of APL (Ctsg-PML-RARA). We identified a Notch expression signature in both human primary APL cells and in Kit+Lin-Sca1+ cells from pre-leukemic Ctsg-PML-RARA mice. Both genetic and pharmacologic inhibition of Notch signaling abrogated the enhanced self-renewal seen in hematopoietic stem/progenitor cells from pre-leukemic Ctsg-PML-RARA mice, but had no influence on cells from age-matched wild-type mice. In addition, six of nine murine APL tumors tested displayed diminished growth in vitro when Notch signaling was inhibited pharmacologically. Finally, we found that genetic inhibition of Notch signaling with a dominant-negative Mastermind-like protein reduced APL growth in vivo in a subset of tumors. These findings expand the role of Notch signaling in hematopoietic diseases, and further define the mechanistic events important for PML-RARA-mediated leukemogenesis.

Although the significance of cathepsin G (CTSG) in host defense has been intensively investigated, little is known about its potential roles in granulopoiesis or leukemogenesis. We report here that CTSG is directly targeted and suppressed by AML1-ETO in t(8;21) acute myeloid leukemia (AML). Luciferase assays demonstrate that the CTSG promoter is strongly transactivated by AML1 and the AML1-dependent transactivation is suppressed by AML1-ETO. We also define a novel regulatory mechanism by which AML1-ETO-mediated transrepression requires both AML1-ETO and AML1 binding at adjacent sites, instead of the replacement of AML1 by AML1-ETO, and wild-type AML1 binding is a prerequisite for the repressive effect caused by AML1-ETO. Further evidence shows that CTSG, as a hematopoietic serine protease, can degrade AML1-ETO both in vitro and in vivo. Restoration of CTSG induces partial differentiation, growth inhibition and apoptosis in AML1-ETO-positive cells. In addition to t(8;21) AML, CTSG downregulation is observed in AML patients with other cytogenetic/genetic abnormalities that potentially interrupt normal AML1 function, that is, inv(16) and EVI1 overexpression. Thus, the targeting and suppression of CTSG by AML1-ETO in t(8;21) AML may provide a mechanism for leukemia cells to escape from the intracellular surveillance system by preventing degradation of foreign proteins.

Infection of host tissues by Staphylococcus aureus and S. epidermidis requires an unusual family of staphylococcal adhesive proteins that contain long stretches of serine-aspartate dipeptide-repeats (SDR). The prototype member of this family is clumping factor A (ClfA), a key virulence factor that mediates adhesion to host tissues by binding to extracellular matrix proteins such as fibrinogen. However, the biological siginificance of the SDR-domain and its implication for pathogenesis remain poorly understood. Here, we identified two novel bacterial glycosyltransferases, SdgA and SdgB, which modify all SDR-proteins in these two bacterial species. Genetic and biochemical data demonstrated that these two glycosyltransferases directly bind and covalently link N-acetylglucosamine (GlcNAc) moieties to the SDR-domain in a step-wise manner, with SdgB appending the sugar residues proximal to the target Ser-Asp repeats, followed by additional modification by SdgA. GlcNAc-modification of SDR-proteins by SdgB creates an immunodominant epitope for highly opsonic human antibodies, which represent up to 1% of total human IgG. Deletion of these glycosyltransferases renders SDR-proteins vulnerable to proteolysis by human neutrophil-derived cathepsin G. Thus, SdgA and SdgB glycosylate staphylococcal SDR-proteins, which protects them against host proteolytic activity, and yet generates major eptopes for the human anti-staphylococcal antibody response, which may represent an ongoing competition between host and pathogen.

Neutrophil elastase (NE) is a neutrophil-derived serine proteinase with broad substrate specificity. We have recently demonstrated that NE is capable of entering tumor cell endosomes and processing novel intracellular substrates. In the current study, we sought to determine the mechanism by which NE enters tumor cells. Our results show that NE enters into early endosomal antigen-1(+) endosomes in a dynamin- and clathrin-dependent but flotillin-1- and caveolin-1-independent fashion. Cathepsin G (but not proteinase-3) also enters tumor endosomes via the same mechanism. We utilized (125)I-labeled NE to demonstrate that NE binds to the surface of cancer cells. Incubation of radiolabeled NE with lung cancer cells displays a dissociation constant (K(d)) of 284 nm. Because NE is known to bind to heparan sulfate- and chondroitin sulfate-containing proteoglycans, we treated cells with glycanases to remove these confounding factors, which did not significantly diminish cell surface binding or endosomal entry. Thus, NE and CG bind to the surface of cancer cells, presumably to a cell surface receptor, and subsequently undergo clathrin pit-mediated endocytosis.

Neutrophil serine proteases play an important role in inflammation by modulating neutrophil effector functions. We have previously shown that neutrophils deficient in the serine proteases cathepsin G and neutrophil elastase (CG/NE neutrophils) exhibit severe defects in chemokine CXCL2 release and reactive oxygen species (ROS) production when activated on immobilized immune complex. Exogenously added active CG rescues these defects, but the mechanism remains undefined. Using a protease-based proteomic approach, we found that, in vitro, the addition of exogenous CG to immune complex-stimulated CG/NE neutrophils led to a decrease in the level of cell-associated annexin A1 (AnxA1) and cathelin-related antimicrobial peptide (CRAMP), both known inflammatory mediators. We further confirmed that, in vivo, CG was required for the extracellular release of AnxA1 and CRAMP in a subcutaneous air pouch model. In vitro, CG efficiently cleaved AnxA1, releasing the active N-terminal peptide Ac2-26, and processed CRAMP in limited fashion. Ac2-26 and CRAMP peptides enhanced the release of CXCL2 by CG/NE neutrophils in a dose-dependent manner via formyl peptide receptor (FPR) stimulation. Blockade of FPRs by an antagonist, Boc2 (t-Boc-Phe-d-Leu-Phe-d-Leu-Phe), abrogates CXCL2 release, whereas addition of FPR agonists, fMLF and F2L, relieves Boc2 inhibition. Furthermore, the addition of active CG, but not inactive CG, also relieves Boc2 inhibition. These findings suggest that CG modulates neutrophil effector functions partly by controlling the release (and proteolysis) of FPR agonists. Unexpectedly, we found that mature CRAMP, but not Ac2-26, induced ROS production through an FPR-independent pathway.

The neutrophil serine proteases cathepsin G (CG) and neutrophil elastase (NE) are involved in immune-regulatory processes and exert antibacterial activity against various pathogens. To date, their role and their therapeutic potential in pulmonary host defense against mycobacterial infections are poorly defined. In this work, we studied the roles of CG and NE in the pulmonary resistance against Mycobacterium bovis bacillus Calmette-Guérin (BCG). CG-deficient mice and even more pronounced CG/NE-deficient mice showed significantly impaired pathogen elimination to infection with M. bovis BCG in comparison to wild-type mice. Moreover, granuloma formation was more pronounced in M. bovis BCG-infected CG/NE-deficient mice in comparison to CG-deficient and wild-type mice. A close examination of professional phagocyte subsets revealed that exclusively neutrophils shuttled CG and NE into the bronchoalveolar space of M. bovis BCG-infected mice. Accordingly, chimeric wild-type mice with a CG/NE-deficient hematopoietic system displayed significantly increased lung bacterial loads in response to M. bovis BCG infection. Therapeutically applied human CG/NE encapsulated in liposomes colocalized with mycobacteria in alveolar macrophages, as assessed by laser scanning and electron microscopy. Importantly, therapy with CG/NE-loaded liposomes significantly reduced mycobacterial loads in the lungs of mice. Together, neutrophil-derived CG and NE critically contribute to deceleration of pathogen replication during the early phase of antimycobacterial responses. In addition, to our knowledge, we show for the first time that liposomal encapsulated CG/NE exhibit therapeutic potential against pulmonary mycobacterial infections. These findings may be relevant for novel adjuvant approaches in the treatment of tuberculosis in humans.

Because PML-RARA-induced acute promyelocytic leukemia (APL) is a morphologically differentiated leukemia, many groups have speculated about whether its leukemic cell of origin is a committed myeloid precursor (e.g. a promyelocyte) versus an hematopoietic stem/progenitor cell (HSPC). We originally targeted PML-RARA expression with CTSG regulatory elements, based on the early observation that this gene was maximally expressed in cells with promyelocyte morphology. Here, we show that both Ctsg, and PML-RARA targeted to the Ctsg locus (in Ctsg-PML-RARA mice), are expressed in the purified KLS cells of these mice (KLS = Kit(+)Lin(-)Sca(+), which are highly enriched for HSPCs), and this expression results in biological effects in multi-lineage competitive repopulation assays. Further, we demonstrate the transcriptional consequences of PML-RARA expression in Ctsg-PML-RARA mice in early myeloid development in other myeloid progenitor compartments [common myeloid progenitors (CMPs) and granulocyte/monocyte progenitors (GMPs)], which have a distinct gene expression signature compared to wild-type (WT) mice. Although PML-RARA is indeed expressed at high levels in the promyelocytes of Ctsg-PML-RARA mice and alters the transcriptional signature of these cells, it does not induce their self-renewal. In sum, these results demonstrate that in the Ctsg-PML-RARA mouse model of APL, PML-RARA is expressed in and affects the function of multipotent progenitor cells. Finally, since PML/Pml is normally expressed in the HSPCs of both humans and mice, and since some human APL samples contain TCR rearrangements and express T lineage genes, we suggest that the very early hematopoietic expression of PML-RARA in this mouse model may closely mimic the physiologic expression pattern of PML-RARA in human APL patients.

Neutrophil serine proteases cathepsin G (CG), neutrophil elastase (NE), and proteinase 3 (PR3) have recently been shown to contribute to killing of Streptococcus pneumoniae in vitro. However, their relevance in lung-protective immunity against different serotypes of S. pneumoniae in vivo has not been determined so far. Here, we examined the effect of CG and CG/NE deficiency on the lung host defense against S. pneumoniae in mice. Despite similar neutrophil recruitment, both CG knockout (KO) mice and CG/NE double-KO mice infected with focal pneumonia-inducing serotype 19 S. pneumoniae demonstrated a severely impaired bacterial clearance, which was accompanied by lack of CG and NE but not PR3 proteolytic activity in recruited neutrophils, as determined using fluorescence resonance energy transfer (FRET) substrates. Moreover, both CG and CG/NE KO mice but not wild-type mice responded with increased lung permeability to infection with S. pneumoniae, resulting in severe respiratory distress and progressive mortality. Both neutrophil depletion and ablation of hematopoietic CG/NE in bone marrow chimeras abolished intra-alveolar CG and NE immunoreactivity and led to bacterial outgrowth in the lungs of mice, thereby identifying recruited neutrophils as the primary cellular source of intra-alveolar CG and NE. This is the first study showing a contribution of neutrophil-derived neutral serine proteases CG and NE to lung-protective immunity against focal pneumonia-inducing serotype 19 S. pneumoniae in mice. These data may be important for the development of novel intervention strategies to improve lung-protective immune mechanisms in critically ill patients suffering from severe pneumococcal pneumonia.

Trousseau syndrome is classically defined as migratory, heparin-sensitive but warfarin-resistant microthrombi in patients with occult, mucinous adenocarcinomas. Injecting carcinoma mucins into mice generates platelet-rich microthrombi dependent on P- and L-selectin but not thrombin. Heparin prevents mucin binding to P- and L-selectin and mucin-induced microthrombi. This model of Trousseau syndrome explains resistance to warfarin, which inhibits fluid-phase coagulation but not selectins. Here we found that carcinoma mucins do not generate microthrombi in mice lacking P-selectin glycoprotein ligand-1 (PSGL-1), the leukocyte ligand for P- and L-selectin. Furthermore, mucins did not activate platelets in blood from PSGL-1-deficient mice. Mucins induced microthrombi in radiation chimeras lacking endothelial P-selectin but not in chimeras lacking platelet P-selectin. Mucins caused leukocytes to release cathepsin G, but only if platelets were present. Mucins failed to generate microthrombi in cathepsin G-deficient mice. Mucins did not activate platelets in blood from mice lacking cathepsin G or protease-activated receptor-4 (PAR4), indicating that cathepsin G activates platelets through PAR4. Using knockout mice and blocking antibodies, we found that mucin-triggered cathepsin G release requires L-selectin and PSGL-1 on neutrophils, P-selectin on platelets, and Src family kinases in both cell types. Thus, carcinoma mucins promote thrombosis through adhesion-dependent, bidirectional signaling in neutrophils and platelets.

Human neutrophil proteinases (elastase, proteinase-3, and cathepsin-G) are released at sites of acute inflammation. We hypothesized that these inflammation-associated proteinases can affect cell signaling by targeting proteinase-activated receptor-2 (PAR(2)). The PAR family of G protein-coupled receptors is triggered by a unique mechanism involving the proteolytic unmasking of an N-terminal self-activating tethered ligand (TL). Proteinases can either activate PAR signaling by unmasking the TL sequence or disarm the receptor for subsequent enzyme activation by cleaving downstream from the TL sequence. We found that none of neutrophil elastase, cathepsin-G, and proteinase-3 can activate G(q)-coupled PAR(2) calcium signaling; but all of these proteinases can disarm PAR(2), releasing the N-terminal TL sequence, thereby preventing G(q)-coupled PAR(2) signaling by trypsin. Interestingly, elastase (but neither cathepsin-G nor proteinase-3) causes a TL-independent PAR(2)-mediated activation of MAPK that, unlike the canonical trypsin activation, does not involve either receptor internalization or recruitment of β-arrestin. Cleavage of synthetic peptides derived from the extracellular N terminus of PAR(2), downstream of the TL sequence, demonstrated distinct proteolytic sites for all three neutrophil-derived enzymes. We conclude that in inflammation, neutrophil proteinases can modulate PAR(2) signaling by preventing/disarming the G(q)/calcium signal pathway and, via elastase, can selectively activate the p44/42 MAPK pathway. Our data illustrate a new mode of PAR regulation that involves biased PAR(2) signaling by neutrophil elastase and a disarming/silencing effect of cathepsin-G and proteinase-3.

Proteinase-activated receptor 1 (PAR(1)) induces activation of platelet and vascular cells after proteolytic cleavage of its extracellular N terminus by thrombin. In pathological situations, other proteinases may be generated in the circulation and might modify the responses of PAR(1) by cleaving extracellular domains. In this study, epitope-tagged wild-type human PAR(1) (hPAR(1)) and a panel of N-linked glycosylation-deficient mutant receptors were permanently expressed in epithelial cells (Kirsten murine sarcoma virus-transformed rat kidney cells and CHO cells). We have analyzed the role of N-linked glycosylation in regulating proteinase activation/disarming and cell global expression of hPAR(1). We reported for the first time that glycosylation in the N terminus of hPAR(1) downstream of the tethered ligand (especially Asn(75)) governs receptor disarming to trypsin, thermolysin, and the neutrophil proteinases elastase and proteinase 3 but not cathepsin G. In addition, hPAR(1) is heavily N-linked glycosylated and sialylated in epithelial cell lines, and glycosylation occurs at all five consensus sites, namely, Asn(35), Asn(62), Asn(75), Asn(250), and Asn(259). Removing these N-linked glycosylation sequons affected hPAR(1) cell surface expression to varying degrees, and N-linked glycosylation at extracellular loop 2 (especially Asn(250)) of hPAR(1) is essential for optimal receptor cell surface expression and receptor stability.

The inhibitory function of tilapia hepcidin (TH)1-5, an antimicrobial peptide, was not examined in previous studies. In this study, we synthesized the TH1-5 peptide and tested TH1-5's antitumor activity against several tumor cell lines. We show that TH1-5 inhibited the proliferation of tumor cells and reduced colony formation in a soft agar assay. Scanning electron microscopy and transmission electron microscopy showed that TH1-5 altered the membrane structure similar to the function of a lytic peptide. Acridine orange/ethidium bromide staining, a wound-healing assay, and a flow cytometric analysis showed that TH1-5 induced necrosis with high-concentration treatment and induced apoptosis with low-concentration treatment. Inflammation is known to be closely associated with the development of cancer. TH1-5 showing anti-inflammatory effects in a previous publication induced us to evaluate the anti-inflammatory effects in cancer cell lines through the expressions of immune-related genes after being treated with the TH1-5 peptide. However, real-time qualitative RT-PCR indicated that TH1-5 treatment induced downregulation of the expressions of interleukin (IL)-6, IL-8, IL-12, IL-15, interferon-γ, CTSG, caspase-7, and Bcl-2, and upregulation of IL-2 and CAPN5 in HeLa cells, and upregulation of IL-8 and CTSG in HT1080 cells. These results suggest that TH1-5 possibly induces an inflammatory response in HeLa cells, but not in HT1080 cells. Overall, these results indicate that TH1-5 possesses the potential to be a novel peptide for cancer therapy., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Cathepsins are involved in regulatory mechanisms in human skin, but their role in photoaged skin remains unknown. This study investigates the role of cathepsin B, D, K, and G in skin photoaging in vivo and in vitro. Cathepsin-induced changes in skin as a result of chronic UV irradiation were detected by immunohistochemistry methods. Protein cathepsin expressions in UVA-induced premature senescence in fibroblasts in vitro were detected by Western blot technique. Cathepsin mRNA expression in photoaged skin and fibroblasts was detected by real-time reverse transcription-polymerase chain reaction. Immunohistochemistry and Western blot show lower protein expression of cathepsin B, D, and K in photoaged skin and fibroblasts, while cathepsin G was higher. The mRNA expression of cathepsin B, D, and K of the photoaged skin in vivo decreased to 20 ± 0.5, 25 ± 1.6 and 22 ± 0.8%, while cathepsin G mRNA increased to 2.24 ± 0.09 times that of control. In photoaged fibroblasts, cathepsin B, D, and K mRNA was downregulated to 64 ± 2.9, 24 ± 2.1 and 9 ± 0.5% while cathepsin G mRNA was upregulated to 1.42 ± 0.06 times that of control fibroblasts. These experiments suggest that cathepsin B, D, K, and G may act as biomarkers in photoaged human skin., (Copyright © 2010 S. Karger AG, Basel.)

NADPH oxidase is a crucial enzyme in mediating antimicrobial host defense and in regulating inflammation. Patients with chronic granulomatous disease, an inherited disorder of NADPH oxidase in which phagocytes are defective in generation of reactive oxidant intermediates (ROIs), suffer from life-threatening bacterial and fungal infections. The mechanisms by which NADPH oxidase mediate host defense are unclear. In addition to ROI generation, neutrophil NADPH oxidase activation is linked to the release of sequestered proteases that are posited to be critical effectors of host defense. To definitively determine the contribution of NADPH oxidase versus neutrophil serine proteases, we evaluated susceptibility to fungal and bacterial infection in mice with engineered disruptions of these pathways. NADPH oxidase-deficient mice (p47(phox-/-)) were highly susceptible to pulmonary infection with Aspergillus fumigatus. In contrast, double knockout neutrophil elastase (NE)(-/-)×cathepsin G (CG)(-/-) mice and lysosomal cysteine protease cathepsin C/dipeptidyl peptidase I (DPPI)-deficient mice that are defective in neutrophil serine protease activation demonstrated no impairment in antifungal host defense. In separate studies of systemic Burkholderia cepacia infection, uniform fatality occurred in p47(phox-/-) mice, whereas NE(-/-)×CG(-/-) mice cleared infection. Together, these results show a critical role for NADPH oxidase in antimicrobial host defense against A. fumigatus and B. cepacia, whereas the proteases we evaluated were dispensable. Our results indicate that NADPH oxidase dependent pathways separate from neutrophil serine protease activation are required for host defense against specific pathogens.