Cathepsin C from extracellular histone-induced M1 alveolar macrophages promotes NETosis during lung ischemia-reperfusion injury.

Primary graft dysfunction (PGD) is a severe form of acute lung injury resulting from lung ischemia/reperfusion injury (I/R) in lung transplantation (LTx), associated with elevated post-transplant morbidity and mortality rates. Neutrophils infiltrating during reperfusion are identified as pivotal contributors to lung I/R injury by releasing excessive neutrophil extracellular traps (NETs) via NETosis. While alveolar macrophages (AMs) are involved in regulating neutrophil chemotaxis and infiltration, their role in NETosis during lung I/R remains inadequately elucidated. Extracellular histones constitute the main structure of NETs and can activate AMs. In this study, we confirmed the significant involvement of extracellular histone-induced M1 phenotype of AMs (M1-AMs) in driving NETosis during lung I/R. Using secretome analysis, public protein databases, and transwell co-culture models of AMs and neutrophils, we identified Cathepsin C (CTSC) derived from AMs as a major mediator in NETosis. Further elucidating the molecular mechanisms, we found that CTSC induced NETosis through a pathway dependent on NADPH oxidase-mediated production of reactive oxygen species (ROS). CTSC could significantly activate p38 MAPK, resulting in the phosphorylation of the NADPH oxidase subunit p47phox, thereby facilitating the trafficking of cytoplasmic subunits to the cell membrane and activating NADPH oxidase. Moreover, CTSC up-regulated and activated its substrate membrane proteinase 3 (mPR3), resulting in an increased release of NETosis-related inflammatory factors. Inhibiting CTSC revealed great potential in mitigating NETosis-related injury during lung I/R. These findings suggests that CTSC from AMs may be a crucial factor in mediating NETosis during lung I/R, and targeting CTSC inhition may represent a novel intervention for PGD in LTx.

Targeting cathepsin C ameliorates murine acetaminophen-induced liver injury.

Acetaminophen (APAP) overdosing is a major cause of acute liver failure worldwide and an established model for drug-induced acute liver injury (ALI). While studying gene expression during murine APAP-induced ALI by 3'mRNA sequencing (massive analysis of cDNA ends, MACE), we observed splenic mRNA accumulation encoding for the neutrophil serine proteases cathepsin G, neutrophil elastase, and proteinase-3 - all are hierarchically activated by cathepsin C (CtsC). This, along with increased serum levels of these proteases in diseased mice, concurs with the established phenomenon of myeloid cell mobilization during APAP intoxication. Objective: In order to functionally characterize CtsC in murine APAP-induced ALI, effects of its genetic or pharmacological inhibition were investigated. Methods and Results: We report on substantially reduced APAP toxicity in CtsC deficient mice. Alleviation of disease was likewise observed by treating mice with the CtsC inhibitor AZD7986, both in short-term prophylactic and therapeutic protocols. This latter observation indicates a mode of action beyond inhibition of granule-associated serine proteases. Protection in CtsC knockout or AZD7986-treated wildtype mice was unrelated to APAP metabolization but, as revealed by MACE, realtime PCR, or ELISA, associated with impaired expression of inflammatory genes with proven pathogenic roles in ALI. Genes consistently downregulated in protocols tested herein included cxcl2, mmp9, and angpt2. Moreover, ptpn22, a positive regulator of the toll-like receptor/interferon-axis, was reduced by targeting CtsC. Conclusions: This work suggests CtsC as promising therapeutic target for the treatment of ALI, among others paradigmatic APAP-induced ALI. Being also currently evaluated in phase III clinical trials for bronchiectasis, successful application of AZD7986 in experimental APAP intoxication emphasizes the translational potential of this latter therapeutic approach.

Toosendanin induces hepatotoxicity by restraining autophagy and lysosomal function through inhibiting STAT3/CTSC axis.

Toosendanin (TSN) is the main active component in the traditional herb Melia toosendan Siebold & Zucc, which exhibits promising potential for development due to its diverse pharmacological properties. However, the hepatotoxicity associated with TSN needs further investigation. Previous research has implicated autophagy dysregulation in TSN-induced hepatotoxicity, yet the underlying mechanisms remain elusive. In this study, the mechanisms of signal transducer and activator of transcription 3 (STAT3) in TSN-induced autophagy inhibition and liver injury were explored using Stat3 knockout C57BL/6 mice and HepG2 cells. TSN decreased cell viability, increased lactate dehydrogenase (LDH) production in vitro, and elevated serum aspartate transaminase (AST) and alanine aminotransferase (ALT) levels as well as liver lesions in vivo, suggesting TSN had significant hepatotoxicity. TSN inhibited Janus kinase 2 (JAK2)/STAT3 pathway and the expression of cathepsin C (CTSC). Inhibition of STAT3 exacerbated TSN-induced autophagy inhibition and hepatic injury, whereas activation of STAT3 attenuated these effects of TSN. Mechanistically, STAT3 transcriptionally regulated the level of CTSC gene, which in turn affected autophagy and the process of liver injury. TSN-administered Stat3 knockout mice showed more severe hepatotoxicity, CTSC downregulation, and autophagy blockade than wildtype mice. In summary, TSN caused hepatotoxicity by inhibiting STAT3/CTSC axis-dependent autophagy and lysosomal function.

CTSC promoted the migration and invasion of glioma cells via activation of STAT3/SERPINA3 axis.

Cathepsin C (CTSC) has been reported to be upregulated in several cancers, however, there are still many missing links about the role of CTSC in glioma. To address this knowledge gap, the present study employed bioinformatics analysis, Transwell assay, RT-qPCR and Western blot assays to investigate the expression level of CTSC in glioma tissues, its relationship with survival period, and its effect on the migration and invasion ability of glioma cells. The findings revealed that CTSC was upregulated in glioma and was associated with poor prognosis. Moreover, CTSC was found to promote cell migration and invasion abilities as well as epithelial-mesenchymal transition (EMT). A further study found that CTSC induced SERPINA3 and STAT3 expression in glioma cells. Additionally, we demonstrated that STAT3 signaling mediated upregulation of SERPINA3 expression by CTSC. In sum, our findings suggest that CTSC activates the STAT3/SERPINA3 axis to promote migration and invasion of glioma cells, which may lead to new potential therapeutic approaches for humans with cancer.

Characterization of a novel dipeptidyl peptidase 1 of Trichinella spiralis and its participation in larval invasion.

The research aimed to describe a new Trichinella spiralis dipeptidyl peptidase 1 (TsDPP1) and investigate its functions in the larval invasion of intestinal epithelial cells (IECs). The gene TsDPP1 was successfully replicated and produced in Escherichia coli BL21 (DE3), showing a strong immune response. TsDPP1 was detected in diverse stages of T. spiralis and showed significant expression in the intestine infective larvae (IIL) and adult worms at 6 days post infection, as confirmed by qPCR and Western blot analysis. The primary localization of TsDPP1 in this parasite was observed in cuticles, stichosomes, and embryos by using the indirect immunofluorescence assay (IIFA). rTsDPP1 exhibited the enzymatic function of natural dipeptidyl peptidase and showed specific binding to IECs, and the binding site was found to be localized on cell membrane. Following transfection with dsRNA-TsDPP1, the expression of TsDPP1 mRNA and protein in muscle larvae (ML) were decreased by approximately 63.52 % and 58.68 %, correspondingly. The activity of TsDPP1 in the ML and IIL treated with dsRNA-TsDPP1 was reduced by 42.98 % and 45.07 %, respectively. The acceleration of larval invasion of IECs was observed with rTsDPP1, while the invasion was suppressed by anti-rTsDPP1 serum. The ability of the larvae treated with dsRNA-TsDPP1 to invade IECs was hindered by 31.23 %. In mice infected with dsRNA-treated ML, the intestinal IIL, and adults experienced a significant decrease in worm burdens and a noticeable reduction in adult female length and fecundity compared to the PBS group. These findings indicated that TsDPP1 significantly impedes the invasion, growth, and reproductive capacity of T. spiralis in intestines, suggesting its potential as a target for anti-Trichinella vaccines.

Different glycosylation profiles of cystatin F alter the cytotoxic potential of natural killer cells.

Cystatin F, a cysteine peptidase inhibitor, is a potent modulator of NK cytotoxicity. By inhibiting granule-mediated cytotoxicity pathway, cystatin F induces formation of non-functional NK cell stage, called split-anergy. We show that N-glycosylation determines the localization and cellular function of cystatin F. Cystatin F mostly exhibited high-mannose glycosylation in U-937 cells, both high-mannose and complex glycosylation in NK-92 and primary NKs, and predominantly complex glycosylation in super-charged NKs. Manipulating N-glycosylation with kifunensine increased high-mannose glycosylation of cystatin F and lysosome localisation, which decreased cathepsin C activity and reduced NK cytotoxicity. Mannose-6-phosphate could significantly reduce the internalization of extracellular cystatin F. By comparing NK cells with different cytotoxic potentials, we found that high-mannose cystatin F was strongly associated with lysosomes and cathepsin C in NK-92 cell line. In contrast, in highly cytotoxic super-charged NKs, cystatin F with complex glycosylation was associated with the secretory pathway and less prone to inhibit cathepsin C. Modulating glycosylation to alter cystatin F localisation could increase the cytotoxicity of NK cells, thereby enhancing their therapeutic potential for treating cancer patients.

Expression and significance of cathepsin C and cathepsin D during pregnancy and Preeclampsia.

BACKGROUND: Cathepsin C (Cat C) is involved in the inflammatory-immune system and can be degraded by cathepsin D (Cat D). Preeclampsia (PE) and the inflammation-immunity relationship is currently a hot research topic, but there are still few studies. The aim was to investigate the expression and significance of Cat C and D in the serum of nonpregnant women, patients in various stages of pregnancy and patients with PE, and in the placenta of patients with normal pregnancy and PE. METHODS: Sixty young healthy nonpregnant women were selected: 180 normal pregnant women, including 60 each in the first, second, and third trimesters, and 100 women with PE, including 39 women with severe preeclampsia. The levels of Cat C and D in serum were detected by enzyme-linked immunosorbent assay (ELISA), and the expression levels of Cat C and D in placentas were detected by immunohistochemistry (IHC). RESULTS: The serum of Cat C in the first trimester was significantly lower than that in the nonpregnant group (P < 0.001), whereas Cat D was significantly higher than that in the nonpregnant group (P < 0.01). The levels of Cat C and D in the second trimester and third trimester were significantly higher than those in the first trimester (P < 0.05), but there was no significant difference in Cat C and D between the second trimester and third trimester. The levels of Cat C in the serum and placentas of patients with PE were significantly higher than those in the third trimester (P < 0.001) and positively correlated with the severity of PE (P < 0.001), whereas the levels of Cat D in the serum and placentas of patients with PE were significantly lower than those in the third trimester (P < 0.001) and negatively correlated with the severity of PE (P < 0.001). Age, primigravida proportion, and body mass index were significantly higher in the PE group than in the control group (P < 0.05), which were high-risk factors for PE. CONCLUSIONS: Cat C and D are associated with the maintenance of normal pregnancy. In patients with preeclampsia, a significant increase in Cat C and a significant decrease in Cat D levels may lead to the occurrence and development of preeclampsia.

E-64c-Hydrazide Based Cathepsin C Inhibitors: Optimizing the Interactions with the S1'-S2' Area.

The zymogens of the neutrophil serine proteases elastase, proteinase 3, and cathepsin G are converted proteolytically into their pro-inflammatory active forms by the action of cathepsin C. The inhibition of this cysteine protease therefore is an interesting therapeutic approach for the treatment of inflammatory disorders with a high neutrophil burden such as COPD. Based on E-64c-hydrazide as lead structure, we have recently developed a covalently acting cathepsin C inhibitor using a n-butyl residue attached at the amine nitrogen of the hydrazide moiety to efficiently address the deep hydrophobic S2 pocket. To further optimize the affinity and selectivity profile of this inhibitor, the S1'-S2' area was now investigated by a combinatorial approach, showing that Nle-tryptamide is a ligand superior to the initially used Leu-isoamylamide. Using the neutrophil precursor line U937 as a cell culture model, this optimized inhibitor blocks the intracellular cathepsin C activity and thereby suppresses the activation of neutrophil elastase.

Abnormal profiles of cathepsin C secreted in urine of Papillon Lefevre syndrome patients.

BACKGROUND: Papillon Lefevre syndrome (PLS) is an autosomal recessive disorder that results from a mutated gene that encodes a lysosomal peptidase known as cathepsin C (CTSC). The clinical presentation of PLS involves mainly palmoplantar keratosis and periodontitis with a variable degree of severity. SUBJECTS: and methods: Our study included ten patients with a broad spectrum of palmoplantar keratosis and periodontitis severity. CTSC variants were detected by Sanger sequencing. CTSC protein secreted in urine was detected by western blotting. RESULTS: Five patients have missense variants, Four have nonsense variants, and one has splice variants in CTSC. The activation products of cathepsin C protein (Heavy and light chains) were absent in all patients' urine samples except one with a significantly reduced level compared to the controls. The dimeric form of CTSC protein was found in all the studied cases. The monomeric form was found in five cases. The products of proteolytic activation of CTSC by other cathepsins (L and S) were found in the urine samples of five of the patients. Each patient had a characteristic pattern of accumulated CTSC protein maturation/activation substrates, intermediates, and products. 40% of the patients had the activation products of other lysosomal cathepsins. CONCLUSION: Urinary CTSC in PLS patients could be used as a diagnostic biomarker for the biochemical screening of the disease. Different variants in CTSC result in different profiles of CTSC secreted in the urine of PLS patients. The profiles of secreted CTSC in urine could be correlated to the severity of palmoplantar keratosis.

Synthesis of Hybrid Tripeptide Peptidomimetics Containing Dehydroamino Acid and Aminophosphonic Acid in the Chain and Evaluation of Their Activity toward Cathepsin C.

Synthesis of a new group of hybrid phosphonodehydropeptides composed of glycyl-(Z)-dehydrophenylalanine and structurally variable aminophosphonates alongside with investigations of their activity towards cathepsin C are presented. Obtained results suggest that the introduction of (Z)-dehydrophenylalanine residue into the short phosphonopeptide chain does induce the ordered conformation. Investigated peptides appeared to act as weak or moderate inhibitors of cathepsin C.

Targeting Cathepsin C in PR3-ANCA Vasculitis.

BACKGROUND: The ANCA autoantigens proteinase 3 (PR3) and myeloperoxidase (MPO) are exclusively expressed by neutrophils and monocytes. ANCA-mediated activation of these cells is the key driver of the vascular injury process in ANCA-associated vasculitis (AAV), and neutrophil serine proteases (NSPs) are disease mediators. Cathepsin C (CatC) from zymogens activates the proteolytic function of NSPs, including PR3. Lack of NSP zymogen activation results in neutrophils with strongly reduced NSP proteins. METHODS: To explore AAV-relevant consequences of blocking NSP zymogen activation by CatC, we used myeloid cells from patients with Papillon-Lefèvre syndrome, a genetic deficiency of CatC, to assess NSPs and NSP-mediated endothelial cell injury. We also examined pharmacologic CatC inhibition in neutrophil-differentiated human hematopoietic stem cells, primary human umbilical vein cells, and primary glomerular microvascular endothelial cells. RESULTS: Patients with Papillon-Lefèvre syndrome showed strongly reduced NSPs in neutrophils and monocytes. Neutrophils from these patients produced a negative PR3-ANCA test, presented less PR3 on the surface of viable and apoptotic cells, and caused significantly less damage in human umbilical vein cells. These findings were recapitulated in human stem cells, in which a highly specific CatC inhibitor, but not prednisolone, reduced NSPs without affecting neutrophil differentiation, reduced membrane PR3, and diminished neutrophil activation upon PR3-ANCA but not MPO-ANCA stimulation. Compared with healthy controls, neutrophils from patients with Papillon-Lefèvre syndrome transferred less proteolytically active NSPs to glomerular microvascular endothelial cells, the cell type targeted in ANCA-induced necrotizing crescentic glomerulonephritis. Finally, both genetic CatC deficiency and pharmacologic inhibition, but not prednisolone, reduced neutrophil-induced glomerular microvascular endothelial cell damage. CONCLUSIONS: These findings may offer encouragement for clinical studies of adjunctive CatC inhibitor in patients with PR3-AAV.

A rare CTSC mutation in Papillon-Lefèvre Syndrome results in abolished serine protease activity and reduced NET formation but otherwise normal neutrophil function.

Papillon-Lefèvre Syndrome (PLS) is an autosomal recessive monogenic disease caused by loss-of-function mutations in the CTSC gene, thus preventing the synthesis of the protease Cathepsin C (CTSC) in a proteolytically active form. CTSC is responsible for the activation of the pro-forms of the neutrophil serine proteases (NSPs; Elastase, Proteinase 3 and Cathepsin G), suggesting its involvement in a variety of neutrophil functions. In PLS neutrophils, the lack of CTSC protease activity leads to inactivity of the NSPs. Clinically, PLS is characterized by an early, typically pre-pubertal, onset of severe periodontal pathology and palmoplantar hyperkeratosis. However, PLS is not considered an immune deficiency as patients do not typically suffer from recurrent and severe (bacterial and fungal) infections. In this study we investigated an unusual CTSC mutation in two siblings with PLS, a 503A>G substitution in exon 4 of the CTSC gene, expected to result in an amino acid replacement from tyrosine to cysteine at position 168 of the CTSC protein. Both patients bearing this mutation presented with pronounced periodontal pathology. The characteristics and functions of neutrophils from patients homozygous for the 503A>G CTSC mutation were compared to another previously described PLS mutation (755A>T), and a small cohort of healthy volunteers. Neutrophil lysates from patients with the 503A>G substitution lacked CTSC protein and did not display any CTSC or NSP activity, yet neutrophil counts, morphology, priming, chemotaxis, radical production, and regulation of apoptosis were without any overt signs of alteration. However, NET formation upon PMA-stimulation was found to be severely depressed, but not abolished, in PLS neutrophils.

Cathepsin C Is Involved in Macrophage M1 Polarization via p38/MAPK Pathway in Sudden Cardiac Death.

This study was aimed at identifying molecular markers associated with the pathogenesis of sudden cardiac death (SCD). It provides a proteomic analysis of human left anterior descending coronary artery from subjects diagnosed with SCD through histological examination and cases of nondisease accidental deaths through autopsy. A total of 2784 proteins were obtained from label-free quantitative proteomic analysis. This included a total of 265 differential proteins which were involved in SCD-related processes, such as inflammation, muscle system process regulation, metal ion transport, and lysosomal pathway. Western blotting was carried out to measure the expressions of cathepsin C (CTSC), focal adhesion kinase (FAK), p-FAK, and proteins related to the p38/MAPK signaling pathway, whereas immunohistochemistry was performed to determine the localization and expression of CTSC, TNF-α, and CD206 in arterial tissues. It was found that CTSC were the most expressed proteins with a significant upward trend in SCD cases. Besides, CTSC regulated macrophage polarization to M1 through the FAK-induced p38/MAPK signaling pathway. This promoted the release of inflammatory factors and eventually increased the inflammatory response. In conclusion, this study implies that CTSC may be one of the key molecular targets for promoting macrophage M1 polarization in SCD, which may provide new therapeutic insights into the treatment of inflammatory diseases.

Cathepsin C inhibition as a potential treatment strategy in cancer.

Epidemiological studies established an association between chronic inflammation and higher risk of cancer. Inhibition of proteolytic enzymes represents a potential treatment strategy for cancer and prevention of cancer metastasis. Cathepsin C (CatC) is a highly conserved lysosomal cysteine dipeptidyl aminopeptidase required for the activation of pro-inflammatory neutrophil serine proteases (NSPs, elastase, proteinase 3, cathepsin G and NSP-4). NSPs are locally released by activated neutrophils in response to pathogens and non-infectious danger signals. Activated neutrophils also release neutrophil extracellular traps (NETs) that are decorated with several neutrophil proteins, including NSPs. NSPs are not only NETs constituents but also play a role in NET formation and release. Although immune cells harbor large amounts of CatC, additional cell sources for this protease exists. Upregulation of CatC expression was observed in different tissues during carcinogenesis and correlated with metastasis and poor patient survival. Recent mechanistic studies indicated an important interaction of tumor-associated CatC, NSPs, and NETs in cancer development and metastasis and suggested CatC as a therapeutic target in a several cancer types. Cancer cell-derived CatC promotes neutrophil recruitment in the inflammatory tumor microenvironment. Because the clinical consequences of genetic CatC deficiency in humans resulting in the elimination of NSPs are mild, small molecule inhibitors of CatC are assumed as safe drugs to reduce the NSP burden. Brensocatib, a nitrile CatC inhibitor is currently tested in a phase 3 clinical trial as a novel anti-inflammatory therapy for patients with bronchiectasis. However, recently developed CatC inhibitors possibly have protective effects beyond inflammation. In this review, we describe the pathophysiological function of CatC and discuss molecular mechanisms substantiating pharmacological CatC inhibition as a potential strategy for cancer treatment.

Cathepsin C inhibitors as anti-inflammatory drug discovery: Challenges and opportunities.

Cathepsin C, an important lysosomal cysteine protease, mediates the maturation process of neutrophil serine proteases, and participates in the inflammation and immune regulation process associated with polymorphonuclear neutrophils. Therefore, cathepsin C is considered to be an attractive target for treating inflammatory diseases. With INS1007 (trade name: brensocatib) being granted a breakthrough drug designation by FDA for the treatment of Adult Non-cystic Fibrosis Bronchiectasis and Coronavirus Disease 2019, the development of cathepsin C inhibitor will attract attentions from medicinal chemists in the future soon. Here, we summarized the research results of cathepsin C as a therapeutic target, focusing on the development of cathepsin C inhibitor, and provided guidance and reference opinions for the upcoming development boom of cathepsin C inhibitor.

Discovery and In Vivo Anti-inflammatory Activity Evaluation of a Novel Non-peptidyl Non-covalent Cathepsin C Inhibitor.

Cathepsin C (Cat C) participates in inflammation and immune regulation by affecting the activation of neutrophil serine proteases (NSPs). Therefore, cathepsin C is an attractive target for treatment of NSP-related inflammatory diseases. Here, the complete discovery process of the first potent "non-peptidyl non-covalent cathepsin C inhibitor" was described with hit finding, structure optimization, and lead discovery. Starting with hit 14, structure-based optimization and structure-activity relationship study were comprehensively carried out, and lead compound 54 was discovered as a potent drug-like cathepsin C inhibitor both in vivo and in vitro. Also, compound 54 (with cathepsin C Enz IC50 = 57.4 nM) exhibited effective anti-inflammatory activity in an animal model of chronic obstructive pulmonary disease. These results confirmed that the non-peptidyl and non-covalent derivative could be used as an effective cathepsin C inhibitor and encouraged us to continue further drug discovery on the basis of this finding.

Human CD4+ T-Cell Clone Expansion Leads to the Expression of the Cysteine Peptidase Inhibitor Cystatin F.

The existence of CD4+ cytotoxic T cells (CTLs) at relatively high levels under different pathological conditions in vivo suggests their role in protective and/or pathogenic immune functions. CD4+ CTLs utilize the fundamental cytotoxic effector mechanisms also utilized by CD8+ CTLs and natural killer cells. During long-term cultivation, CD4+ T cells were also shown to acquire cytotoxic functions. In this study, CD4+ human T-cell clones derived from activated peripheral blood lymphocytes of healthy young adults were examined for the expression of cytotoxic machinery components. Cystatin F is a protein inhibitor of cysteine cathepsins, synthesized by CD8+ CTLs and natural killer cells. Cystatin F affects the cytotoxic efficacy of these cells by inhibiting the major progranzyme convertases cathepsins C and H as well as cathepsin L, which is involved in perforin activation. Here, we show that human CD4+ T-cell clones express the cysteine cathepsins that are involved in the activation of granzymes and perforin. CD4+ T-cell clones contained both the inactive, dimeric form as well as the active, monomeric form of cystatin F. As in CD8+ CTLs, cysteine cathepsins C and H were the major targets of cystatin F in CD4+ T-cell clones. Furthermore, CD4+ T-cell clones expressed the active forms of perforin and granzymes A and B. The levels of the cystatin F decreased with time in culture concomitantly with an increase in the activities of granzymes A and B. Therefore, our results suggest that cystatin F plays a role in regulating CD4+ T cell cytotoxicity. Since cystatin F can be secreted and taken up by bystander cells, our results suggest that CD4+ CTLs may also be involved in regulating immune responses through cystatin F secretion.

Causative role of mast cell and mast cell-regulatory function of disialyllacto-N-tetraose in necrotizing enterocolitis.

Necrotizing enterocolitis (NEC) remains a fatal gastrointestinal disorder in neonates. Disialyllacto-N-tetraose (DSLNT), a function-unclear human milk-derived hexasaccharide, shows anti-NEC potential in previous animal studies. This study is aimed to explore the role of mast cell (MC), a fundamental cell type of mucosal immune system and protective DSLNT in regulating pathological process of NEC. For this purpose, infantile intestinal-tissues were collected from NEC neonates for examination of MCs and its proteases-positive cells. MC accumulation and MC-specific proteases (chymase, tryptase and dipeptidyl peptidase I) were firstly found in lesioned area of NEC infants in-vivo. Subsequent in-situ experiments on neonatal ileum segments showed that purified MC-chymase induced a destructive epithelial layer shedding from basement and microvascular endothelium damage in infantile intestinal segments. Human foreskin MC-activation model was established and DSLNT were applied; MC products (histamine and MC-proteases) were used as MC activation/degranulation indicators. In this in-vitro model, DSLNT pretreatment suppressed release of histamine, chymase and tryptase by MC to the tissue supernatants during lipopolysaccharide or complement C5a stimulation. Newborn rats were formula-hand-fed with or without DSLNT supplement and exposed to hypoxia/cold-stress to induce experimental-NEC-model. In NEC rats, DSLNT supplementation reduced the incidence and pathological scores of NEC, inhibited local accumulation of MC and reduced cytokines (IL-1ß, IL-6 and TNF-α) levels in the ileum of rats. In conclusion, MC was causally implicated in epithelium barrier failure in pathogenesis of NEC. DSLNT favorably modulated MC homeostasis by regulating MC degranulation/accumulation, contributing to attenuated NEC. This indicated novel pathomechanisms and potential targets of NEC.

Cathepsin C promotes breast cancer lung metastasis by modulating neutrophil infiltration and neutrophil extracellular trap formation.

Lung metastasis is the major cause of breast cancer-related mortality. The neutrophil-associated inflammatory microenvironment aids tumor cells in metastatic colonization in lungs. Here, we show that tumor-secreted protease cathepsin C (CTSC) promotes breast-to-lung metastasis by regulating recruitment of neutrophils and formation of neutrophil extracellular traps (NETs). CTSC enzymatically activates neutrophil membrane-bound proteinase 3 (PR3) to facilitate interleukin-1ß (IL-1ß) processing and nuclear factor κB activation, thus upregulating IL-6 and CCL3 for neutrophil recruitment. In addition, the CTSC-PR3-IL-1ß axis induces neutrophil reactive oxygen species production and formation of NETs, which degrade thrombospondin-1 and support metastatic growth of cancer cells in the lungs. CTSC expression and secretion are associated with NET formation and lung metastasis in human breast tumors. Importantly, targeting CTSC with compound AZD7986 effectively suppresses lung metastasis of breast cancer in a mouse model. Overall, our findings reveal a mechanism of how tumor cells regulate neutrophils in metastatic niches and support CTSC-targeting approaches for cancer treatment.

The CTSC-RAB38 Fusion Transcript Is Associated With the Risk of Hemorrhage in Brain Arteriovenous Malformations.

Brain arteriovenous malformations (bAVMs) are congenital anomalies of blood vessels that cause intracranial hemorrhage in children and young adults. Chromosomal rearrangements and fusion genes play an important role in tumor pathogenesis, though the role of fusion genes in bAVM pathophysiological processes is unclear. The aim of this study was to identify fusion transcripts in bAVMs and analyze their effects. To identify fusion transcripts associated with bAVM, RNA sequencing was performed on 73 samples, including 66 bAVM and 7 normal cerebrovascular samples, followed by STAR-Fusion analysis. Reverse transcription polymerase chain reaction and Sanger sequencing were applied to verify fusion transcripts. Functional pathway analysis was performed to identify potential effects of different fusion types. A total of 21 fusion transcripts were detected. Cathepsin C (CTSC)-Ras-Related Protein Rab-38 (RAB38) was the most common fusion and was detected in 10 of 66 (15%) bAVM samples. In CTSC-RAB38 fusion-positive samples, CTSC and RAB38 expression was significantly increased and activated immune/inflammatory signaling. Clinically, CTSC-RAB38 fusion bAVM cases had a higher hemorrhage rate than non-CTSC-RAB38 bAVM cases (p < 0.05). Our study identified recurrent CTSC-RAB38 fusion transcripts in bAVMs, which may be associated with bAVM hemorrhage by promoting immune/inflammatory signaling.