Repression of CTSG, ELANE and PRTN3-mediated histone H3 proteolytic cleavage promotes monocyte-to-macrophage differentiation.

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.

Simultaneous inhibition of two neutrophil serine proteases by the S. aureus innate immune evasion protein EapH2.

Extracellular adherence protein domain (EAP) proteins are high-affinity, selective inhibitors of neutrophil serine proteases (NSP), including cathepsin-G (CG) and neutrophil elastase (NE). Most Staphylococcus aureus isolates encode for two EAPs, EapH1 and EapH2, that contain a single functional domain and share 43% identity with one another. Although structure/function investigations from our group have shown that EapH1 uses a globally similar binding mode to inhibit CG and NE, NSP inhibition by EapH2 is incompletely understood due to a lack of NSP/EapH2 cocrystal structures. To address this limitation, we further studied NSP inhibition by EapH2 in comparison with EapH1. Like its effects on NE, we found that EapH2 is a reversible, time-dependent, and low nanomolar affinity inhibitor of CG. We characterized an EapH2 mutant which suggested that the CG binding mode of EapH2 is comparable to EapH1. To test this directly, we used NMR chemical shift perturbation to study EapH1 and EapH2 binding to CG and NE in solution. Although we found that overlapping regions of EapH1 and EapH2 were involved in CG binding, we found that altogether distinct regions of EapH1 and EapH2 experienced changes upon binding to NE. An important implication of this observation is that EapH2 might be capable of binding and inhibiting CG and NE simultaneously. We confirmed this unexpected feature by solving crystal structures of the CG/EapH2/NE complex and demonstrating their functional relevance through enzyme inhibition assays. Together, our work defines a new mechanism of simultaneous inhibition of two serine proteases by a single EAP protein.

Characterization of two distinct neutrophil serine protease-binding modes within a Staphylococcus aureus innate immune evasion protein family.

Extracellular adherence protein domain (EAPs) proteins are a class of innate immune evasion proteins secreted by the human pathogen Staphylococcus aureus. EAPs are potent and selective inhibitors of cathepsin-G (CG) and neutrophil elastase (NE), which are the two most abundant neutrophil serine proteases (NSPs). Previous work from our group has shown that the prototypical EAP, EapH1, relies on plasticity within a single inhibitory site to block the activities of CG and NE. However, whether other EAPs follow similar structure-function relationships is unclear. To address this question, we studied the inhibitory properties of the first (Eap1) and second (Eap2) domains of the modular extracellular adherence protein of S. aureus and determined their structures when bound to CG and NE, respectively. We observed that both Eap1 and Eap2 displayed time-dependent inhibition of CG (on the order of 10-9 M) and of NE (on the order of 10-10 M). We also found that whereas the structures of Eap1 and Eap2 bound to CG showed an overall inhibitory mode like that seen previously for EapH1, the structures of Eap1 and Eap2 bound to NE revealed a new inhibitory mode involving a distal region of the EAP domain. Using site-directed mutagenesis of Eap1 and Eap2, along with enzyme assays, we confirmed the roles of interfacial residues in NSP inhibition. Taken together, our work demonstrates that EAPs can form structurally divergent complexes with two closely related serine proteases and further suggests that certain EAPs may be capable of inhibiting two NSPs simultaneously.

Immunoregulatory and neutrophil-like monocyte subsets with distinct single-cell transcriptomic signatures emerge following brain injury.

Monocytes represent key cellular elements that contribute to the neurological sequela following brain injury. The current study reveals that trauma induces the augmented release of a transcriptionally distinct CD115+/Ly6Chi monocyte population into the circulation of mice pre-exposed to clodronate depletion conditions. This phenomenon correlates with tissue protection, blood-brain barrier stability, and cerebral blood flow improvement. Uniquely, this shifted the innate immune cell profile in the cortical milieu and reduced the expression of pro-inflammatory Il6, IL1r1, MCP-1, Cxcl1, and Ccl3 cytokines. Monocytes that emerged under these conditions displayed a morphological and gene profile consistent with a subset commonly seen during emergency monopoiesis. Single-cell RNA sequencing delineated distinct clusters of monocytes and revealed a key transcriptional signature of Ly6Chi monocytes enriched for Apoe and chitinase-like protein 3 (Chil3/Ym1), commonly expressed in pro-resolving immunoregulatory monocytes, as well as granule genes Elane, Prtn3, MPO, and Ctsg unique to neutrophil-like monocytes. The predominate shift in cell clusters included subsets with low expression of transcription factors involved in monocyte conversion, Pou2f2, Na4a1, and a robust enrichment of genes in the oxidative phosphorylation pathway which favors an anti-inflammatory phenotype. Transfer of this monocyte assemblage into brain-injured recipient mice demonstrated their direct role in neuroprotection. These findings reveal a multifaceted innate immune response to brain injury and suggest targeting surrogate monocyte subsets may foster tissue protection in the brain.

PAD4-Induced NETosis Via Cathepsin G-Mediated Platelet-Neutrophil Interaction in ChAdOx1 Vaccine-Induced Thrombosis-Brief Report.

BACKGROUND: Vaccine-induced immune thrombotic thrombocytopenia (VITT) is a rare syndrome characterized by platelet anti-PF4 (platelet-activating antiplatelet factor 4)-related thrombosis. Platelet-neutrophil interaction has been suggested to play a role, but the underlying mechanism has not been fully elucidated. METHODS: The study included 10 patients with VITT after ChAdOx1 (chimpanzee adenovirus Oxford 1) nCoV-19 (Oxford-AstraZeneca) vaccine administration, 10 patients with ischemic stroke (IS), 10 patients with acute deep vein thrombosis, and 10 control subjects in whom blood levels of neutrophil extracellular traps (NETs), soluble TF (tissue factor), and thrombin generation were examined. Furthermore, we performed in vitro studies comparing the effect of serum from patients and controls on NETs formation. Finally, immunohistochemistry was performed in cerebral thrombi retrieved from a patients with VITT and 3 patients with IS. RESULTS: Compared with patients with IS, patients with deep vein thrombosis, controls, and patients with VITT had significantly higher blood values of CitH3 (citrullinated histone H3), soluble TF, D-dimer, and prothrombin fragment 1+2 (P<0.0001). Blood CitH3 significantly correlated with blood soluble TF (Spearman rank correlation coefficient=0.7295; P=0.0206) and prothrombin fragment 1+2 (Spearman rank correlation coefficient=0.6809; P<0.0350) in patients with VITT. Platelet-neutrophil mixture added with VITT plasma resulted in higher NETs formation, soluble TF and thrombin generation, and platelet-dependent thrombus growth under laminar flow compared with IS and deep vein thrombosis plasma; these effects were blunted by PAD4 (protein arginine deiminase 4) and cathepsin G inhibitors, anti-FcγRIIa (Fc receptor for IgG class IIa), and high doses of heparin. Immunohistochemistry analysis showed a more marked expression of PAD4 along with more diffuse neutrophil infiltration and NETs formation as well as TF and cathepsin expression in VITT thrombus compared with thrombi from patients with IS. CONCLUSIONS: Patients with VITT display enhanced thrombogenesis by PAD4-mediated NETs formation via cathepsin G-mediated platelet/neutrophil interaction.

Cathepsin G promotes arteriovenous fistula maturation by positively regulating the MMP2/MMP9 pathway.

BACKGROUND: Arteriovenous fistula (AVF) is currently the preferred vascular access for hemodialysis patients. However, the low maturation rate of AVF severely affects its use in patients. A more comprehensive understanding and study of the mechanisms of AVF maturation is urgently needed. METHODS AND RESULTS: In this study, we downloaded the publicly available datasets (GSE119296 and GSE220796) from the Gene Expression Omnibus (GEO) and merged them for subsequent analysis. We screened 84 differentially expressed genes (DEGs) and performed the functional enrichment analysis. Next, we integrated the results obtained from the degree algorithm provided by the Cytohubba plug-in, Molecular complex detection (MCODE) plug-in, weighted gene correlation network analysis (WGCNA), and Least absolute shrinkage and selection operator (LASSO) logistic regression. This integration allowed us to identify CTSG as a hub gene associated with AVF maturation. Through the literature search and Pearson's correlation analysis, the genes matrix metalloproteinase 2 (MMP2) and MMP9 were identified as potential downstream effectors of CTSG. We then collected three immature clinical AVF vein samples and three mature samples and validated the expression of CTSG using immunohistochemistry (IHC) and double-immunofluorescence staining. The IHC results demonstrated a significant decrease in CTSG expression levels in the immature AVF vein samples compared to the mature samples. The results of double-immunofluorescence staining revealed that CTSG was expressed in both the intima and media of AVF veins. Moreover, the expression of CTSG in vascular smooth muscle cells (VSMCs) was significantly higher in the mature samples compared to the immature samples. The results of Masson's trichrome and collagen I IHC staining demonstrated a higher extent of collagen deposition in the media of immature AVF veins compared to the mature. By constructing an in vitro CTSG overexpression model in VSMCs, we found that CTSG upregulated the expression of MMP2 and MMP9 while downregulating the expression of collagen I and collagen III. Furthermore, CTSG was found to inhibit VSMC migration. CONCLUSIONS: CTSG may promote AVF maturation by stimulating the secretion of MMP2 and MMP9 from VSMCs and reducing the extent of medial fibrosis in AVF veins by inhibiting the secretion of collagen I and collagen III.

Targeted Library of Phosphonic-Type Inhibitors of Human Neutrophil Elastase.

Despite many years of research, human neutrophil elastase (HNE) still remains an area of interest for many researchers. This multifunctional representative of neutrophil serine proteases is one of the most destructive enzymes found in the human body which can degrade most of the extracellular matrix. Overexpression or dysregulation of HNE may lead to the development of several inflammatory diseases. Previously, we presented the HNE inhibitor with kinact/KI value over 2,000,000 [M-1s-1]. In order to optimize its structure, over 100 novel tripeptidyl derivatives of α-aminoalkylphosphonate diaryl esters were synthesized, and their activity toward HNE was checked. To confirm the selectivity of the resultant compounds, several of the most active were additionally checked against the two other neutrophil proteases: proteinase 3 and cathepsin G. The developed modifications allowed us to obtain a compound with significantly increased inhibitory activity against human neutrophil elastase with high selectivity toward cathepsin G, but none toward proteinase 3.

Association of admission neutrophil serine proteinases levels with the outcomes of acute ischemic stroke: a prospective cohort study.

BACKGROUND: Neutrophil serine proteinases (NSPs), released by activated neutrophils, are key proteins involved in the pathophysiologic processes of stroke. NSPs are also implicated in the process and response of thrombolysis. This study aimed to analyze three NSPs (neutrophil elastase, cathepsin G, and proteinase 3) in relation to acute ischemic stroke (AIS) outcomes and in relation to the outcomes of patients treated with intravenous recombinant tissue plasminogen activator (IV-rtPA). METHODS: Among 736 patients prospectively recruited at the stroke center from 2018 to 2019, 342 patients diagnosed with confirmed AIS were included. Plasma neutrophil elastase (NE), cathepsin G (CTSG), and proteinase 3 (PR3) concentrations were measured on admission. The primary endpoint was unfavorable outcome defined as modified Rankin Scale score 3-6 at 3 months, and the secondary endpoints were symptomatic intracerebral hemorrhage (sICH) within 48 h, and mortality within 3 months. In the subgroup of patients who received IV-rtPA, post-thrombolysis early neurological improvement (ENI) (defined as National Institutes of Health Stroke Scale score = 0 or decrease of ≥ 4 within 24 h after thrombolysis) was also included as the secondary endpoint. Univariate and multivariate logistic regression analyses were performed to evaluate the association between NSPs levels and AIS outcomes. RESULTS: Higher NE and PR3 plasma levels were associated with the 3-month mortality and 3-month unfavorable outcome. Higher NE plasma levels were also associated with the risk of sICH after AIS. After adjusting for potential confounders, plasma NE level > 229.56 ng/mL (odds ratio [OR] = 4.478 [2.344-8.554]) and PR3 > 388.77 ng/mL (OR = 2.805 [1.504-5.231]) independently predicted the 3-month unfavorable outcome. Regarding rtPA treatment, patients with NE plasma concentration > 177.22 ng/mL (OR = 8.931 [2.330-34.238]) or PR3 > 388.77 ng/mL (OR = 4.275 [1.045-17.491]) were over 4 times more likely to suffer unfavorable outcomes after rtPA treatment. The addition of NE and PR3 to clinical predictors of unfavorable functional outcome after AIS and the outcome after rtPA treatment improved discrimination as well as reclassification (integrated discrimination improvement = 8.2% and 18.1%, continuous net reclassification improvement = 100.0% and 91.8%, respectively). CONCLUSIONS: Plasma NE and PR3 are novel and independent predictors of 3-month functional outcomes after AIS. Plasma NE and PR3 also possess predictive value to identify patients with unfavorable outcomes after rtPA treatment. NE is probably an important mediator of the effects of neutrophils on stroke outcomes, which worth further investigation.

Newly revealed variants of SERPINA3 in generalized pustular psoriasis attenuate inhibition of ACT on cathepsin G.

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.

Vitamin D supplementation induces CatG-mediated CD4+ T cell inactivation and restores pancreatic ß-cell function in mice with type 1 diabetes.

Type 1 diabetes (T1D) is a chronic autoimmune disease accompanied by the immune-mediated destruction of pancreatic ß-cells. In this study, we aimed to explore the regulatory effects of vitamin D (VD) supplementation on pancreatic ß-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 ß-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 ß-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 ß-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 ß-cell function in T1D.NEW & NOTEWORTHY We report that vitamin D (VD) supplementation downregulates CatG expression and inhibits CD4+ T cell activation, thereby improving ß-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.

Identification and validation of two alternatively spliced novel isoforms of human α-1-antichymotrypsin.

α-1-antichymotrypsin (ACT) is a serine proteinase inhibitor that controls the activity of proteases like chymotrypsin, cathepsin G and mast cell chymase. Familial variants of ACT results in liver and lung diseases, but it is also reported to be associated with several other disease conditions. ACT is mainly synthesized in the liver using four coding exons, namely E1, E2, E3 and E4 encoding a 423 amino acid protein that also includes a 23 amino acid signal peptide. It is found to be associated with amyloid plaques and is elevated during inflammatory response and modulates cytokine based signal transduction pathways, independent of its anti-protease activity. Therefore, the multispecificity of ACT and its non-inhibitory roles in diseased conditions warrants an assessment of possible existence of the other isoforms. Consequently, scanning of introns, 5' and 3' region of the ACT gene using computational tools like FGENESH and FEX did indicate the presence of coding regions. Using a combined approach of bioinformatics and molecular biology, we have found one novel exon located in the intronic region between exons E1 and E2, that splices with exon E2 and replaces N-terminal exon E1, generating an ACT isoform with a novel 151 base pair N-terminus. This isoform was found to lack the signal sequence and is smaller in size but its reactive centre loop remains intact. A truncated transcript was also confirmed with an extension of the E3 by a 12 nucleotide intronic region including a stop codon. Modelling studies show that due to removal of E4 this isoform lacks the RCL. Novel isoform ACT-N lacks E1 but has a conserved RCL. However, due to loss of strands of ß-sheet A, it may also be inactive, but with ability to bind the target proteases. The novel truncated ACT-T isoform lacks the RCL and may have a non-inhibitory role. These hypothesis will need further work for functional validation.

The expression of glycosyltransferases sdgA and sdgB in Staphylococcus epidermidis depends on the conditions of biofilm formation.

The Staphylococcus aureus SdrG protein is glycosylated by SdgA and SdgB for protection against its degradation by the neutrophil cathepsin G. So far, there is no information about the role of Staphylococcus epidermidis SdgA or SdgB in biofilm-forming; therefore, the focus of this work was to determine the distribution and expression of the sdrG, sdgA and sdgB genes in S. epidermidis under in vitro and in vivo biofilm conditions. The frequencies of the sdrG, sdgA and sdgB genes were evaluated by PCR in a collection of 75 isolates. Isolates were grown in dynamic (non-biofilm-forming) or static (biofilm-forming) conditions. The expression of sdrG, sdgA and sdgB was determined by RT-qPCR in cells grown under dynamic conditions (CGDC), as well as in planktonic and sessile cells from a biofilm and cells adhered to a catheter implanted in Balb/c mice. The sdrG and sdgB genes were detected in 100% of isolates, while the sdgA gene was detected in 71% of the sample (p < 0.001). CGDC did not express sdrG, sdgA and sdgB mRNAs. Planktonic and sessile cells expressed sdrG and sdgB, and the same was observed in cells adhered to the catheter. In particular, one isolate, capable of inducing a biofilm under treatment with cathepsin G, expressed sdrG and sdgB in planktonic and sessile cells and cells adhering to the catheter. This suggests that bacteria require biofilm conditions as an important factor for the transcription of the sdgA, sdgB and sdrG genes.

INSIGHTS INTO THE REGULATION OF MAST CELL FUNCTION IN TYPE 2 INFLAMMATION.

Type 2 inflammation (T2I) underlies the pathogenesis of asthma, chronic rhinosinusitis with nasal polyps, and eosinophilic esophagitis. Mast cells (MCs) are tissue resident hematopoietic effector cells thought to play major roles in T2I. Two subtypes of human MCs are recognized based on immunohistochemical differences. MCs expressing tryptase but not chymase (MCT) reside within mucosal epithelial surfaces, and MCs expressing tryptase, chymase, and cathepsin G (MCTC) reside in submucosal, perivascular and intraneural locations. During T2I, MCs (particularly MCT) increase markedly by unclear mechanisms. Single cell genomic studies reveal that traditional histochemical categorization vastly underestimates the extent of MC functional heterogeneity. MCT and MCTC likely reflect endpoints of a developmental continuum, emerging from a transitional stage of development in which MCs expand through in situ proliferation. This mechanism, likely driven by interleukin 4 and other cytokines, is unique among granulocytes and carries substantial implications for pathogenesis and therapy of T2I-associated diseases.

Cathepsin G-Induced Cell Aggregation of Breast Cancer MCF-7 Decreases Doxorubicin Sensitivity in a Hypoxia-Inducible Factor-Independent Mechanism.

Solid tumors habitually harbor regions with insufficient oxygen away from vasculature. Hypoxia is an important factor that confers malignant phenotypes like chemoresistance to tumor cells. We have demonstrated that cathepsin G (CG) stimulates cell aggregation in breast cancer MCF-7 cells by activating insulin-like growth factor-1 signaling. We investigated whether cancer cell aggregates induced by CG acquire hypoxia-dependent chemoresistance. Pimonidazole staining and hypoxia-inducible factor (HIF)-1α and -2α expression indicated that the core of the cell aggregates was hypoxic. Electrophoretic mobility shift and reporter assays showed that the CG-induced cell aggregates displayed transcriptional activity through HIF-responsive elements. Moreover, HIF target genes PGK1 and SLC2A1 demonstrated upregulated expression in CG-induced cell aggregates, indicating that the aggregates expressed functional HIF. Doxorubicin (DXR)-induced cytotoxicity was significantly lower in the cell aggregates induced by CG compared with monolayer cells under normoxia. Unexpectedly, the upregulation of P-glycoprotein expression, which is reported to be a HIF-target gene, and decreasing intracellular accumulation of DXR was not detected in the cell aggregates as opposed to in monolayer cells under normoxia. Additionally, reduction of DXR sensitivity in the aggregates was not suppressed by treatment with the HIF inhibitor, YC-1 and HIF-1α small interfering RNA (siRNA). Therefore, we conclude that cell aggregation induced by CG decreases DXR sensitivity via a HIF-independent mechanism.

Low Concentration of the Neutrophil Proteases Cathepsin G, Cathepsin B, Proteinase-3 and Metalloproteinase-9 Induce Biofilm Formation in Non-Biofilm-Forming Staphylococcus epidermidis Isolates.

Neutrophils play a crucial role in eliminating bacteria that invade the human body; however, cathepsin G can induce biofilm formation in a non-biofilm-forming Staphylococcus epidermidis 1457 strain, suggesting that neutrophil proteases may be involved in biofilm formation. Cathepsin G, cathepsin B, proteinase-3, and metalloproteinase-9 (MMP-9) from neutrophils were tested on the biofilm induction in commensal (skin isolated) and clinical non-biofilm-forming S. epidermidis isolates. From 81 isolates, 53 (74%) were aap+, icaA−, icaD− genotype, and without the capacity of biofilm formation under conditions of 1% glucose, 4% ethanol or 4% NaCl, but these 53 non-biofilm-forming isolates induced biofilm by the use of different neutrophil proteases. Of these, 62.3% induced biofilm with proteinase-3, 15% with cathepsin G, 10% with cathepsin B and 5% with MMP -9, where most of the protease-induced biofilm isolates were commensal strains (skin). In the biofilm formation kinetics analysis, the addition of phenylmethylsulfonyl fluoride (PMSF; a proteinase-3 inhibitor) showed that proteinase-3 participates in the cell aggregation stage of biofilm formation. A biofilm induced with proteinase-3 and DNAse-treated significantly reduced biofilm formation at an early time (initial adhesion stage of biofilm formation) compared to untreated proteinase-3-induced biofilm (p < 0.05). A catheter inoculated with a commensal (skin) non-biofilm-forming S. epidermidis isolate treated with proteinase-3 and another one without the enzyme were inserted into the back of a mouse. After 7 days of incubation period, the catheters were recovered and the number of grown bacteria was quantified, finding a higher amount of adhered proteinase-3-treated bacteria in the catheter than non-proteinase-3-treated bacteria (p < 0.05). Commensal non-biofilm-forming S. epidermidis in the presence of neutrophil cells significantly induced the biofilm formation when multiplicity of infection (MOI) 1:0.01 (neutrophil:bacteria) was used, but the addition of a cocktail of protease inhibitors impeded biofilm formation. A neutrophil:bacteria assay did not induce neutrophil extracellular traps (NETs). Our results suggest that neutrophils, in the presence of commensal non-biofilm-forming S. epidermidis, do not generate NETs formation. The effect of neutrophils is the production of proteases, and proteinase-3 releases bacterial DNA at the initial adhesion, favoring cell aggregation and subsequently leading to biofilm formation.

Neutrophil Cathepsin G Enhances Thrombogenicity of Mildly Injured Arteries via ADP-Mediated Platelet Sensitization.

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.

Variation of Proteolytic Cleavage Sites towards the N-Terminal End of the S2 Subunit of the Novel SARS-CoV-2 Omicron Sublineage BA.2.12.1.

The prevalence of novel SARS-CoV-2 variants is also accompanied by an increased turnover rate and additional cleavage sites at the positions necessary for priming the Spike (S) protein. Of these priming sites, the proteolytically sensitive polybasic sequence of the activation loop at the S1/S2 interface and the S2' location within the S2 subunit of the S protein are cleaved by furin and TMPRSS2, which are important for the infection of the target cell. Neutrophils, migrating to the site of infection, secrete serine proteases to fight against pathogens. The serine proteases encompass neutrophil elastase (NE), proteinase 3 (PR3), and cathepsin G (CatG), which can hydrolyze the peptide bond adjacent to the S1/S2 interface. SARS-CoV-2 might take the opportunity to hijack proteases from an immune response to support viral entry to the cell. The region near S704L within the S2 subunit, a novel amino acid substitution of SARS-CoV-2 Omicron sublineage BA.2.12.1, is located close to the S1/S2 interface. We found that NE, PR3, and CatG digested the peptide within this region; however, the S704L amino acid substitution altered cleavage sites for PR3. In conclusion, such an amino acid substitution modifies S2 antigen processing and might further impact the major histocompatibility complex (MHC) binding and T cell activation.

Trivalent chromium supplementation ameliorates adjuvant induced rheumatoid arthritis through up-regulation of FOXP3 and decrease in synovial Cathepsin G expression.

BACKGROUND: Rheumatoid arthritis (RA) is a known debilitating autoimmune disease. Immune-suppressants that are used for disease treatment have serious side effects, therefore, trivalent chromium (Cr (III)); which has shown evidence of its influences on some inflammatory pathways and cytokines; was used in this study for the first time to be assessed for its therapeutic effect in RA rat model and was compared to prednisolone in a trial to find a treatment with lesser side effects. METHODS: Adult male albino rats were randomly divided into four groups: normal, untreated RA, prednisolone treated RA (1.25 mg/kg/day) and Cr (III) treated RA groups (80 µg/kg/day), induction of RA was done by subcutaneous complete Freund adjuvant injection. Study duration was 4 weeks throughout which arthritis scoring and weight measurement were pursued. Histopathological examination and immunohistochemical FOXP3 assessment were done for joint biopsies. Serum inflammatory markers (interleukin 17, interleukin 10, CRP) and synovial erosive arthritis marker (Cathepsin G) were measured. HDL and non-HDL cholesterol were estimated as well. RESULTS: Cr (III) treatment showed marked clinical and histopathological improvement, also astonishing anti-inflammatory effects (increase in FOXP3 expression and interleukin 10, with decrease in interleukin 17, CRP and synovial Cathepsin G) to the extent that Cr (III) effects on inflammation abolishment were comparable to that of prednisolone and even better at some aspects. Moreover, Cr (III) was protective from side effects, i.e., weight gain and dyslipidemia that were seen with prednisolone treatment. CONCLUSIONS: Cr (III) is promising in treating RA and it lacks some side effects of accustomed immune-modulatory agents including prednisolone. Further experimental studies and clinical trials should be held to see the efficacy of Cr (III) in different doses and to assess its long term side effects when used for rheumatoid arthritis and other autoimmune diseases treatment.

Cathepsin G Degrades Staphylococcus aureus Biofilms.

Polymorphonuclear leukocytes (PMN) phagocytose and kill individual bacteria but are far less efficient when challenged with bacterial aggregates. Consequently, growth within a biofilm affords Staphylococcus aureus some protection but PMN penetrate S. aureus biofilms and phagocytose bacteria, suggesting that enzymes released through neutrophil degranulation degrade biofilms into fragments small enough for phagocytosis. Here we show that the capacity of PMN to invade biofilms depended largely on the activity of secreted cathepsin G.

Local structural plasticity of the Staphylococcus aureus evasion protein EapH1 enables engagement with multiple neutrophil serine proteases.

Members of the EAP family of Staphylococcus aureus immune evasion proteins potently inhibit the neutrophil serine proteases (NSPs) neutrophil elastase, cathepsin-G, and proteinase-3. Previously, we determined a 1.8 Å resolution crystal structure of the EAP family member EapH1 bound to neutrophil elastase. This structure revealed that EapH1 blocks access to the enzyme's active site by forming a noncovalent complex with this host protease. To determine how EapH1 inhibits other NSPs, we studied here the effects of EapH1 on cathepsin-G. We found that EapH1 inhibits cathepsin-G with a Ki of 9.8 ± 4.7 nm Although this Ki value is ∼466-fold weaker than the Ki for EapH1 inhibition of neutrophil elastase, the time dependence of inhibition was maintained. To define the physical basis for EapH1's inhibition of cathepsin-G, we crystallized EapH1 bound to this protease, solved the structure at 1.6 Å resolution, and refined the model to Rwork and Rfree values of 17.4% and 20.9%, respectively. This structure revealed a protease-binding mode for EapH1 with cathepsin-G that was globally similar to that seen in the previously determined EapH1-neutrophil elastase structure. The nature of the intermolecular interactions formed by EapH1 with cathepsin-G differed considerably from that with neutrophil elastase, however, with far greater contributions from the inhibitor backbone in the cathepsin-G-bound form. Together, these results reveal that EapH1's ability to form high-affinity interactions with multiple NSP targets is due to its remarkable level of local structural plasticity.