Release Of Cathepsin Research Articles

PF206 DUAL TARGETING OF LYSOSOMES AND MITOCHONDRIA BY ANTIHISTAMINES TO ERADICATE LEUKEMIC CELLS

Background:A number of different genetic alterations are known to be involved in leukemogenesis but, to date, only MLL fusion genes have been demonstrated capable of independently inducing the transformation of hematopoietic progenitors and mature cells into AML cells in vivo. Early transformation‐related events are considered to induce general alterations in cell physiology that lay the foundations for cancer cell progression. We then hypothesized that reverting those early leukemogenic changes would potentially undermine pathways that ultimately sustain AML, and performed an in silico screening to identify drugs of interest.Aims:The main objective of the study was to reposition drugs against AML based on a transformation‐associated gene signature and characterize its mechanism of action.Methods:For the in silico screening, an early MLL‐AF9 gene signature was queried in connectivity maps, and drugs were ranked according to their potential to revert the gene signature. To study the antileukemic effects of selected drugs, cell viability and clonogenicity studies were performed in 4 human AML cell lines, cells from 9 AML patients, cells from 5 healthy‐donor buffy coats and 4 cord blood samples. In vivo studies were performed with conditioned NOD.Cg‐Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice xenotransplanted with AML or cord blood cells treated with antihistamines for 18 h and engrafted for 8 weeks. Effects on lysosomal compartment were assessed by Lysotracker staining, galectin puncta formation and cathepsin B immunofluorescence studies. Effects on mitochondria were studied by rhodamine‐123 and MitoSOX staining, and with caspaseGlo 3/7 assay.Results:Antihistamine drug family was identified in the screening as reverting the MLL‐AF9‐associated gene signature, and, indeed, 4 out of 10 tested antihistamines (ebastine, loratadine, terfenadine, rupatadine) proved effective against AML in vitro and ex vivo in the low micromolar range, while mostly sparing healthy cells. Antileukemic effects were observed irrespective of specific AML subtypes and mutations. In vivo studies further corroborated the results, with ebastine treatment practically abolishing AML engraftment while sparing healthy hematopoiesis. When the mechanism of action was interrogated, effects were shown to be histamine receptor 1‐independent, as no reversion was observed with the agonists for the receptor and several highly‐specific antihistamines had no effect. Moreover, cytotoxic effects were observed in cell lines with no expression of histamine receptor 1. Further studies concerning the mechanism of action found an expansion in lysosomes followed by lysosomal membrane permeabilization and cathepsin release to cytoplasm, described to lead to cell death. Concomitantly to lysosomal expansion, we detected dissipation of mitochondrial membrane potential, mitochondrial reactive oxygen species generation and caspase activation upon antihistamine treatment. The 4 antileukemic antihistamines shared cationic amphiphilic properties, known to induce tropism to both targeted organelles thus providing an explanation for the observed effects.Summary/Conclusion:A group of antihistamines possessing cationic amphiphilic structure selectively eradicate AML cells independently of histamine receptors by targeting both lysosomes and mitochondria, known to be altered and more fragile in AML compared to their healthy counterpart. This dual mechanism is based on physicochemical properties of drugs and emerges as a novel therapeutic approach for AML eradication.

Stunning stress-induced textural softening in silver carp (Hypophthalmichthys molitrix) fillets and underlying mechanisms

This study aimed to investigate the effect of stunning stress on textural properties and water-holding capacity (WHC) of silver carp (Hypophthalmichthys molitrix) fillets and its underlying mechanisms based on intrinsic proteases activity, myofibril microstructure, and myowater dynamic measurements. The results suggested that compared with the non-stressful percussion, ice/water and gill cut stunning resulted in accelerated muscle tenderization with intensive myofibril disintegration and sarcolemma-myofibril detachments. Myowater mobility and distribution were altered by stunning stress, in which gill cut facilitated the migration of immobilized water and free water, thus enhancing the centrifugation loss. In addition, stunning stress promoted the activation of endogenous proteases and the release of cathepsin B and L from lysosomes to myofibrils, which showed significant (P < 0.01) correlations with decreased shear force and elevated myofibril fragmentation index of fillets. Therefore, faster accumulation of cathepsins and myofibril disintegration together contributed to the weakening of texture and WHC in stunning-stressed fish fillets.

Cytotoxin 1 from Naja atra Cantor venom induced necroptosis of leukemia cells

BackgroundCytotoxin 1 (CTX1) purified from Naja atra Cantor venom could inhibit cancer cell proliferation, but the mechanism is not clear. This study aimed to investigate the mechanism by which leukemia cells are killed by CTX1. Materials and methodsHL-60 and KG1a cells were treated with CTX1 and the cell death was detected. ResultsThe viability of HL-60 and KG1a cells decreased in a dose- and time-dependent manner after treatment with CTX1. CTX1 mainly induced late apoptosis and necrosis. The cell death induced by CTX1 could be rescued by specific necroptosis inhibitor Nec-1 but not by caspase inhibitor Z-VAD-fmk in HL-60 cells. In addition, CTX1 increased lysosome membrane permeability (LMP) and release of cathepsin B. ConclusionCTX1 could induce necroptosis in leukemia cells, and it is related to LMP increase and cathepsin release. CTX1 could be a promising anti-cancer drug for leukemia therapy.

Brucella abortus nitric oxide metabolite regulates inflammasome activation and IL-1β secretion in murine macrophages.

NLRP3 inflammasome is a protein complex crucial to caspase-1 activation and IL-1β and IL-18 maturation. This receptor participates in innate immune responses to different pathogens, including the bacteria of genus Brucella. Our group recently demonstrated that Brucella abortus-induced IL-1β secretion involves NLRP3 inflammasome and it is partially dependent on mitochondrial ROS production. However, other factors could be involved, such as P2X7-dependent potassium efflux, membrane destabilization, and cathepsin release. Moreover, there is increasing evidence that nitric oxide acts as a modulator of NLRP3 inflammasome. The aim of this study was to unravel the mechanism of NLRP3 inflammasome activation induced by B. abortus, as well as the involvement of bacterial nitric oxide (NO) as a modulator of this inflammasome pathway. We demonstrated that NO produced by B. abortus can be used by the bacteria to modulate IL-1β secretion in infected murine macrophages. Additionally, our results suggest that B. abortus-induced IL-1β secretion depends on a P2X7-independent potassium efflux, lysosomal acidification, cathepsin release, mechanisms clearly associated to NLRP3 inflammasome. In summary, our results help to elucidate the molecular mechanisms of NLRP3 activation and regulation during an intracellular bacterial infection.

HLA class II antibodies induce necrotic cell death in human endothelial cells via a lysosomal membrane permeabilization-mediated pathway

Antibody-mediated rejection (AMR) is the major cause of allograft loss after solid organ transplantation. Circulating donor-specific antibodies against human leukocyte antigen (HLA), in particular HLA class II antibodies are critical for the pathogenesis of AMR via interactions with endothelial cells (ECs). To investigate the effects of HLA class II antibody ligation to the graft endothelium, a model of HLA-DR antibody-dependent stimulation was utilized in primary human ECs. Antibody ligation of HLA class II molecules in interferon-γ-treated ECs caused necrotic cell death without complement via a pathway that was independent of apoptosis and necroptosis. HLA-DR-mediated cell death was blocked by specific neutralization of antibody ligation with recombinant HLA class II protein and by lentiviral knockdown of HLA-DR in ECs. Importantly, HLA class II-mediated cytotoxicity was also induced by relevant native allele-specific antibodies from human allosera. Necrosis of ECs in response to HLA-DR ligation was mediated via hyperactivation of lysosomes, lysosomal membrane permeabilization (LMP), and release of cathepsins. Notably, LMP was caused by reorganization of the actin cytoskeleton. This was indicated by the finding that LMP and actin stress fiber formation by HLA-DR antibodies were both downregulated by the actin polymerization inhibitor cytochalasin D and inhibition of Rho GTPases, respectively. Finally, HLA-DR-dependent actin stress fiber formation and LMP led to mitochondrial stress, which was revealed by decreased mitochondrial membrane potential and generation of reactive oxygen species in ECs. Taken together, ligation of HLA class II antibodies to ECs induces necrotic cell death independent of apoptosis and necroptosis via a LMP-mediated pathway. These findings may enable novel therapeutic approaches for the treatment of AMR in solid organ transplantation.

Organometallic ruthenium and iridium phosphorus complexes: Synthesis, cellular imaging, organelle targeting and anticancer applications

The use of metal complexes containing phosphorus ligands as anticancer agents has not been well studied. In this work, eight novel half‐sandwich IrIII and RuII compounds with P^P‐chelating ligands have been synthesized and fully characterized, and alongside two crystal structures were reported. All eight complexes displayed highly potent antiproliferative activity, up to nine times more potent than the clinical anticancer drug cisplatin towards A549 lung cancer cells. Complex Ir1, which has a simpler structure and highly potent antiproliferative activity, was selected to investigate in further mechanistic studies. No hydrolysis and nucleobase binding occurred for complex Ir1. In order to elucidate subcellular localization, the self‐luminescence of the complex Ir1 was utilized. Ir1 can specifically target lysosomes and facilitate excessive production of reactive oxygen species, resulting in lysosomal membrane permeabilization in A549 cells. Release of cathepsin B and changes in the mitochondria membrane potential also contributed to the observed cytotoxicity of Ir1, which demonstrated an anticancer action mechanism that was different from that of cisplatin. The favorable results from biological and chemical research demonstrated that these types of complexes hold significant theranostic potential.

Harmine induced apoptosis in Spodoptera frugiperda Sf9 cells by activating the endogenous apoptotic pathways and inhibiting DNA topoisomerase I activity

Harmine, a useful botanical compound, has demonstrated insecticidal activity against some pests. However, harmine's mechanism of action has not been thoroughly elucidated to date. To preliminarily explore harmine's insecticidal mechanisms, the cytotoxicity of harmine against Spodoptera frugiperda Sf9 cells was comprehensively investigated. Our results indicated that harmine induced apoptosis in Sf9 cells, as evidenced by cellular and nuclear morphological changes, DNA laddering and increases in caspase-3-like activities. In addition, activation of the mitochondrial apoptotic pathway by harmine was confirmed by the generation of ROS, opening of mitochondrial permeability transition pores (MPTPs), increase in cytosolic Ca2+, changes in mRNA expression levels of genes involved in the mitochondrial apoptotic pathway and increase and release of Cytochrome c. Furthermore, lysosomal membrane permeabilization, release of cathepsin L from the lysosome into the cytosol and cleavage of caspase-3 were also triggered, which indicated that lysosomes were involved in this physiological process. Moreover, the effect of harmine on DNA topoisomerase I activity was investigated by in vivo and molecular docking experiments. These data not only verified that harmine induced apoptosis via comprehensive activation of the mitochondrial and lysosomal pathways and inhibition of DNA topoisomerase I activity in Sf9 cells but also revealed a mechanism of harmine insecticidal functions for pest control.

Clomiphene citrate induces nuclear translocation of the TFEB transcription factor and triggers apoptosis by enhancing lysosomal membrane permeabilization

The autophagy-lysosome pathway plays a central role in cellular homeostasis by regulating the cellular degradative machinery. The transcription factor EB (TFEB) regulates the biogenesis and function of both lysosomes and autophagosomes, and enhancement of TFEB function has emerged as an attractive therapeutic strategy for lysosome-related disorders. However, little is known about the role of TFEB activation in regulating the cellular fate. Here, we describe that clomiphene citrate (CC), a selective estrogen receptor modulator, promotes nuclear translocation of TFEB and increases lysosomal biogenesis in HeLa and MDA-MB-231 cells. Treatment with CC inhibits cell viability and causes apoptosis by increasing the release of proteases cathepsin B (CatB) and cathepsin D (CatD) from lysosomes into the cytosol. In contrast, knockdown of TFEB rescues the cells from CC-induced cell death. Furthermore, CC-induced TFEB activation also enhances the autophagy flux in HeLa cells. Knockdown of autophagy-related gene 7 (ATG7) significantly decreases the CC-induced CatB and CatD release and cell death, suggesting that autophagy contributes to the lysosomal membrane permeabilization (LMP) caused by CC. Altogether, these findings have broad implications for our understanding of TFEB function and provide new insights into CC pharmacological therapy.

CID1067700, a late endosome GTPase Rab7 receptor antagonist, attenuates brain atrophy, improves neurologic deficits and inhibits reactive astrogliosis in rat ischemic stroke.

Increasing evidence suggests that Ras-related in brain 7 (Rab7), an endosome-localized small GTPase contributes to cerebral ischemic brain injury. In the present study, we investigated the role of Rab7 in ischemic stroke-induced formation of astrogliosis and glial scar. Rats were subjected to transient middle cerebral artery occlusion (tMCAO); the rats were injected with the Rab7 receptor antagonist CID1067700 (CID). Primary astrocytes were subjected to an oxygen and glucose deprivation and reoxygenation (OGD/Re) procedure; CID was added to the cell culture media. We found that Rab7 was significantly elevated over time in both the in vivo and in vitro astrocytic injury models, and administration of CID significantly down-regulated the glial scar markers such as glial fibillary acidic protein (GFAP), neurocan and phosphacan. Moreover, administration of CID significantly attenuated the brain atrophy and improved neurologic deficits in tMCAO rats, and protected astrocytes against OGD/Re-induced injury. Further, CID downregulated the protein levels of Lamp1 and active cathepsin B in astrocytes after OGD/Re or tMCAO injury; CID inhibited the co-localization of cathepsin B and Rab7, Lamp1 and Rab7; CID decreased OGD/Re-induced increase in lysosomal membrane permeability and blocked OGD/Re-induced release of cathepsin B from the lysosome into the cytoplasm in astrocytes. Taken together, these results suggest that Rab7 is involved in ischemic stroke-induced formation of astrogliosis and glial scar. CID administration attenuates brain atrophy and improves neurologic deficits and inhibits astrogliosis and glial scar formation after ischemic stroke via reducing the activation and release of cathepsin B from the lysosome into the cytoplasm.

Regulated Necrosis Orchestrates Microglial Cell Death in Manganese-Induced Toxicity

Microglia, the brain resident immune cells, play prominent roles in immune surveillance, tissue repair and neural regeneration. Despite these pro-survival actions, the relevance of these cells in the progression of several neuropathologies has been established. In the context of manganese (Mn) overexposure, it has been proposed that microglial activation contributes to enhance the neurotoxicity. However, the occurrence of a direct cytotoxic effect of Mn on microglial cells remains controversial. In the present work, we investigated the potential vulnerability of immortalized mouse microglial cells (BV-2) toward Mn2+, focusing on the signaling pathways involved in cell death. Evidence obtained showed that Mn2+ induces a decrease in cell viability which is associated with reactive oxygen species (ROS) generation. In this report we demonstrated, for the first time, that Mn2+ triggers regulated necrosis (RN) in BV-2 cells involving two central mechanisms: parthanatos and lysosomal disruption. The occurrence of parthanatos is supported by several cellular and molecular events: (i) DNA damage; (ii) AIF translocation from mitochondria to the nucleus; (iii) mitochondrial membrane permeabilization; and (iv) PARP1-dependent cell death. On the other hand, Mn2+ induces lysosomal membrane permeabilization (LMP) and cathepsin D (CatD) release into the cytosol supporting the lysosomal disruption. Pre-incubation with CatB and D inhibitors partially prevented the Mn2+-induced cell viability decrease. Altogether these events point to lysosomes as players in the execution of RN. In summary, our results suggest that microglial cells could be direct targets of Mn2+ damage. In this scenario, Mn2+ triggers cell death involving RN pathways.

Treponema pallidum promotes macrophage polarization and activates the NLRP3 inflammasome pathway to induce interleukin-1\u03b2 production

BackgroundThe involvement of inflammasome activation and macrophage polarization during the process of syphilis infection remains unknown. In this study, A series of experiments were performed using human macrophages to research the role of NLRP3 inflammasome regulation in interleukin (IL)-1β production and its influence on macrophage polarization triggered by T. pallidum.ResultsThe results showed that in M0 macrophages treated with T. pallidum, the M1-associated markers inducible nitric oxide synthase (iNOS), IL-1β and TNF-α were upregulated, and the M2-associated markers CD206 and IL-10 were downregulated. In addition, we observed NLRP3 inflammasome activation and IL-1β secretion in T. pallidum-treated macrophages, and the observed production of IL-1β occurred in a dose- and time-dependent manner. Moreover, the secretion of IL-1β by macrophages after T. pallidum treatment was notably reduced by anti-NLRP3 siRNA and caspase-1 inhibitor treatment. NAC, KCl, and CA074-ME treatment also suppressed IL-1β release from T. pallidum-treated macrophages.ConclusionsThese findings showed that T. pallidum induces M0 macrophages to undergo M1 macrophage polarization and elevate IL-1β secretion through NLRP3. Moreover, the process of NLRP3 inflammasome activation and IL-1β production in macrophages in response to T. pallidum infection involves K+ efflux, mitochondrial ROS production and cathepsin release. This study provides a new insight into the innate immune response to T. pallidum infection.

Lysosomal permeabilization and endoplasmic reticulum stress mediate the apoptotic response induced after photoactivation of a lipophilic zinc(II) phthalocyanine.

We have previously reported that the phototoxic action of the lipophilic phthalocyanine Pc9 (2,9(10),16(17),23(24) tetrakis[(2-dimethylamino)ethylsulfanyl]phthalocyaninatozinc(II)) encapsulated into poloxamine micelles is related to the induction of an apoptotic response in murine colon CT26 carcinoma cells. In the present study, we explored the intracellular signals contributing to the resulting apoptotic death. We found that Pc9-T1107 arrests cell cycle progression immediately after irradiation promoting then an apoptotic response. Thus, 3 h after irradiation the percentage of hypodiploid cells increased from 5.9 ± 0.6% to 23.1 ± 0.1%; activation of caspases 8 and 9 was evident; the population of cells with loss of mitochondrial membrane potential increased from 1.1 ± 0.4% to 44.0 ± 9.3%; the full-length forms of Bid and PARP-1 were cleaved; and a 50% decrease of the expression levels of the anti-apoptotic proteins Bcl-2 and Bcl-XL was detected. We also found that the photosensitizer, mainly retained in lysosomes and endoplasmic reticulum (ER), promotes the permeabilization of lysosomal membranes and induces ER stress. Lysosomal membrane permeabilization was demonstrated by the reduction of acridine orange lysosome fluorescence, the release of Cathepsin D into the cytosol and ∼50% decrease of Hsp70, a chaperone recognized as a lysosomal stabilizer. Cathepsin D also contributed to Bid cleavage and caspase 8 activation. The oxidative damage to the ER induced an unfolded protein response characterized, 3 h after irradiation, by a 3-fold increase in cytosolic Ca2+ levels and 3-4 times higher expression of ER chaperones GRP78/BIP, calnexin, Hsp90 and Hsp110. The cell death signaling promoted by cytosolic Ca2+, calpains and lysosomal proteases was partially abolished by the Ca2+ chelator BAPTA-AM, the calpain inhibitor PD 150606 and proteases inhibitors. Furthermore, Bax down-regulation observed in Pc9-treated cells was undetectable in the presence of PD 150606, indicating that calpains contribute to Bax proteolytic damage. In summary, our results indicate that photoactivation of Pc9-T1107 led to lysosomal membrane permeabilization, induction of ER stress and activation of a caspase-dependent apoptotic cell death.

Transcription factor E3 protects against cadmium-induced apoptosis by maintaining the lysosomal-mitochondrial axis but not autophagic flux in Neuro-2a cells

Cadmium (Cd), is a well-known environmental and occupational hazard with a potent neurotoxic action. However, the mechanism underlying cadmium-induced neurotoxicity remains unclear. Herein, we exposed Neuro-2a cells to different concentrations of cadmium chloride (CdCl2) (12.5, 25 and 50 μM) for 24 h and found that Cd significantly induced lysosomal membrane permeabilization (LMP) with the release of cathepsin B (CTSB) to the cytosol, which in turn caused the release of mitochondrial cytochrome c (Cyt c) and eventually triggered caspase-dependent apoptosis. Interestingly, Cd decreased TFE3 expression but induced the nuclear translocation of TFE3 and TFE3 target-gene expression, which might be associated with lysosomal stress mediated by Cd. Notably, Tfe3 overexpression protected against Cd-induced neurotoxicity by maintaining the lysosomal-mitochondrial axis, and the protective effect of TFE3 is not dependent on the restoration of autophagic flux. In conclusion, our study demonstrated for the first time that lysosomal-mitochondrial axis dependent apoptosis, a neglected mechanism, may be the most important reason for Cd-induced neurotoxicity and that manipulation of TFE3 signaling may be a potential therapeutic approach for treatment of Cd-induced neurotoxicity.

FasL on the surface of Tag7 (PGRP-S)-activated lymphocytes induces necroptosis in HLA-negative tumor cells with the involvement of lysosomes and mitochondria

Recently we have found that cytokine IL-2 and innate immunity protein Tag7 activate cytotoxic lymphocytes that kill HLA-negative tumor cells, inducing both apoptosis and necroptosis. Here we decrypt the processes, taking part in necroptosis execution after FasL-Fas interaction. Necroptosis begins with RIPK1 activation and necrosome formation. Subsequent activation of MLKL results in the increase of Ca2+ level in the cell and activation of Ca2+-dependent enzymes causing lysosomal membrane permeabilization and the release of cathepsins to the cytosol. STAT3 translocation to the mitochondria and binding to a component of the respiratory chain complex I causes ROS accumulation. We have shown that transduction of necroptotic signal via TNFR1 and Fas has many common points. It is known that apoptosis plays a major role in physiological cell death; however, under pathological conditions necroptosis is very common. That is why the detailed mechanisms of FasL-Fas necroptosis can help in understanding the processes of elimination of tumor cells that have blocked apoptosis signal transduction.

ROS-mediated lysosomal membrane permeabilization is involved in bupivacaine-induced death of rabbit intervertebral disc cells

Bupivacaine is frequently administered for diagnosing and controlling spine-related pain in interventional spine procedures. However, the potential cytotoxic effects of bupivacaine on intervertebral disc (IVD) cells and the underlying molecular mechanisms have not yet been fully established. Here, we showed that bupivacaine decreased the viability of rabbit IVD cells in a dose- and time-dependent manner. Moreover, the short-term cytotoxicity of bupivacaine in IVD cells was primarily due to cell necrosis, as assessed by Annexin V-propidium iodide staining and live/dead cell staining. Necrosis was verified by observations of swollen organelles, plasma membrane rupture, and cellular lysis under transmission electronic microscopy. Interestingly, our data indicated that bupivacaine-induced primary necrosis might involve the necroptosis pathway. The key finding of this study was that bupivacaine was able to induce lysosomal membrane permeabilization (LMP) with the release of cathepsins into the cytosol, as evidenced by LysoTracker Red staining, acridine orange staining, and cathepsin D immunofluorescence staining. Consistently, inhibitors of lysosomal cathepsins, CA074-Me and pepstatin A, significantly reduced bupivacaine-induced cell death. Finally, we found that bupivacaine resulted in an increase in intracellular reactive oxygen species (ROS) and that inhibition of ROS by N-acetyl-L-cysteine effectively blocked bupivacaine-induced LMP and cell death. In summary, the results of this in vitro study reveal a novel mechanism underlying bupivacaine-induced cell death involving ROS-mediated LMP. Our findings establish a basis for the further investigation of bupivacaine cytotoxicity in an in vivo system.

Lysosomal Cathepsin Release Is Required for NLRP3-Inflammasome Activation by Mycobacterium tuberculosis in Infected Macrophages.

Lysosomal cathepsin B (CTSB) has been proposed to play a role in the induction of acute inflammation. We hypothesised that the presence of active CTSB in the cytosol is crucial for NLRP3-inflammasome assembly and, consequently, for mature IL-1β generation after mycobacterial infection in vitro. Elevated levels of CTSB was observed in the lungs of mice and rabbits following infection with Mycobacterium tuberculosis (Mtb) H37Rv as well as in plasma from acute tuberculosis patients. H37Rv-infected murine bone marrow-derived macrophages (BMDMs) displayed both lysosomal leakage, with release of CTSB into the cytosol, as well as increased levels of mature IL-1β. These responses were diminished in BMDM infected with a mutant H37Rv deficient in ESAT-6 expression. Pharmacological inhibition of cathepsin activity with CA074-Me resulted in a substantial reduction of both mature IL-1β production and caspase-1 activation in infected macrophages. Moreover, cathepsin inhibition abolished the interaction between NLRP3 and ASC, measured by immunofluorescence imaging in H37Rv-infected macrophages, demonstrating a critical role of the enzyme in NLRP3-inflammasome activation. These observations suggest that during Mtb infection, lysosomal release of activated CTSB and possibly other cathepsins inhibitable by CA07-Me is critical for the induction of inflammasome-mediated IL-1β processing by regulating NLRP3-inflammasome assembly in the cytosol.

Neutrophil cathepsin G inhibits hapten-presenting dendritic cell production of IL-12 and skewing of hapten-reactive CD4 T Cell development to IFN-γ- and IL-17-producing effector cells in allergic contact sensitivity

Abstract Contact hypersensitivity (CHS) is a CD8 T cell-mediated response to hapten skin sensitization and challenge. Sensitization of WT mice induces hapten-reactive CD8 cells producing CHS effector cytokines IFN-γ/IL-17 and IL-4-producing CD4 cells that cannot mediate CHS. Although CXCR2-dependent Gr-1+ cell recruitment into hapten challenged skin is required to direct effector CD8 T cell infiltration to elicit CHS, sensitization of CXCR2−/− mice or neutrophil-depleted WT mice induced both hapten-reactive CD4 and CD8 cells producing IFN-γ and IL-17. CD4 T cell-mediated CHS responses were not generated during hapten sensitization of neutrophil depleted WT mice treated with anti-IL-12 mAb or neutrophil depleted IL-12−/−mice. Neutrophil depletion during hapten sensitization of WT mice markedly increased numbers of IL-12-producing hapten-primed dendritic cells (DC), DC-derived p40, and T-bet-expressing CD4 T cells in the skin-draining lymph nodes. Sensitization of mice lacking the neutrophil serine protease cathepsin G induced hapten-reactive CD4 and CD8 T cells producing IFN-γ and IL-17 with elevated and elongated CHS responses to challenge. Few Gr-1+ cells were detected in the skin draining lymph nodes following senstization suggesting this regulation may not be mediated through direct Gr-1-DC interactions. In support of this, induction of CHS effector CD4 T cells producing IFN-γ in neutrophil-depleted WT mice was eliminated by subcutaneous injection of active, but not inactivated, cathepsin G during sensitization. Thus, hapten skin sensitization induces neutrophil release of cathepsin G that systemically inhibits hapten-presenting dendritic cell production of IL-12 and the development of CHS effector CD4 T cells.

Macrophage digesting dysfunction by Angiotensin II through lysosomal cathepsin B‐mediated Nlrp3 inflammasome activation

Angiotensin II (Ang II) is an octapeptide hormone that plays a significant role in mediating hypertension. Hypertension is considered a chronic inflammatory disease; however, the molecular basis underlying the sterile inflammatory response involved in the process of hypertension remains unclear. The present study aims to investigate the role of macrophage Nlrp3 inflammasomes in engulfing and digesting microbes, which are associated with macrophage function, an early onset of hypertension‐associated macrophage injury. Our findings demonstrate that AngII stimulation decreased the NO release and macrophage digestion in cultured RAW264.7 cell. Accompanied with these changes, AngII markedly increased the Nlrp3 inflammasome formation and activation. As AngII‐induced up‐regulation of Nlrp3 inflammasome in macrophage was attributed to lysosome dysfunction induced cathepsin B release. In addition, Losartan, a nonpeptide Ang II receptor antagonist, decreased AngII‐induced Nlrp3 inflammasome activation, lysosomal membrane permeability, lysosomal cathepsin B release, and macrophage digestion dysfunction in macrophage. Similarly, AngII induced macrophage digestion microbes and NO production, which were blocked by Nlrp3 gene silencing and ATI gene silencing. It is concluded that Angiotensin II enhanced lysosomal membrane permeabilization and consequent release of lysosomal cathepsin B, resulting in activation of the macrophage Nlrp3 inflammasome, which may contribute to NO mediated the development of digesting microbes dysfunction.Support or Funding InformationThis study was supported by the National Natural Science Foundation of China (81603587 and 81603668) and by the Guangdong Science and Technology Department (2017A020211016).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Defect-related luminescent bur-like hydroxyapatite microspheres induced apoptosis of MC3T3-E1 cells by lysosomal and mitochondrial pathways.

When orthopedic joints coated by hydroxyapatite (HA) were implanted in the human body, they release wear debris into the surrounding tissues. The generation and accumulation of wear particles will induce aseptic loosening. However, the potential bioeffect and mechanism of HA-coated orthopedic implants on bone cells are poorly understood. In this study, defect-related luminescent bur-like hydroxyapatite (BHA) microspheres with the average diameter of 7-9 μm which are comparable to that of the wear-debris particles from aseptically loosened HA implants or HA debris have been synthesized by hydrothermal synthesis and the MC3T3-E1 cells were set as a cells model to study the potential bioeffect and mechanism of BHA microspheres. The studies demonstrated that BHA microspheres could be taken into MC3T3-E1 cells via endocytosis involved in micropinocytosis- and clathrin-mediated endocytosis process, and exert cytotoxicity effect. BHA microspheres could induce the cell apoptosis by intracellular production of reactive oxygen species (ROS), which led to not only an increase in the permeability of lysosome and release of cathepsins B, but also mitochondrial dysfunction and DNA damage. Our results provide novel evidence to elucidate their toxicity mechanisms and might be helpful for more reasonable applications of HA-based orthopaedic implants in the future.

Cathepsin B-Mediated NLRP3 Inflammasome Formation and Activation in Angiotensin II -Induced Hypertensive Mice: Role of Macrophage Digestion Dysfunction

Background/Aims: Angiotensin II (Ang II) is an octapeptide hormone that plays a significant role in mediating hypertension. Although hypertension is considered a chronic inflammatory disease, the molecular basis of the sterile inflammatory response involved in hypertension remains unclear. Methods: We investigated the role of macrophage NLRP3 inflammasomes in engulfing and digesting microbes, a key macrophage function, and in early onset of hypertension-associated macrophage injury using biochemical analyses, gene silencing, molecular biotechnology, immunofluorescence, and microbiology. Results: Ang II stimulation decreased nitric oxide (NO) release and macrophage digestion in cultured THP-1 cells and markedly increased NLRP3 inflammasome formation and activation. NO release and macrophage digestion were restored by NLRP3 inflammasome inhibition with isoliquiritigenin and gene silencing. This Ang II-induced upregulation of NLRP3 inflammasomes in macrophages was attributed to lysosomal damage and release of cathepsin B. Mechanistically, losartan, a nonpeptide Ang II receptor antagonist, decreased Ang II-induced NLRP3 inflammasome activation, lysosomal membrane permeability, lysosomal cathepsin B release, and macrophage digestion dysfunction. Similarly, Ang II-induced macrophage microbe digestion and NO production, which were blocked by ATI gene silencing. In addition, in vivo experiments showed that the bacteria scavenging function was clearly decreased in macrophages from Ang II-induced hypertensive mice. Conclusion: Angiotensin II enhances lysosomal membrane permeabilization and the consequent release of lysosomal cathepsin B, resulting in activation of the macrophage NLRP3 inflammasome. This may contribute to NO mediation of dysfunction in digesting microbes.