Airway epithelial CD47 plays a critical role in inducing influenza virus-mediated bacterial super-infection.

Respiratory viral infection increases host susceptibility to secondary bacterial infections, yet the precise dynamics within airway epithelia remain elusive. Here, we elucidate the pivotal role of CD47 in the airway epithelium during bacterial super-infection. We demonstrated that upon influenza virus infection, CD47 expression was upregulated and localized on the apical surface of ciliated cells within primary human nasal or bronchial epithelial cells. This induced CD47 exposure provided attachment sites for Staphylococcus aureus, thereby compromising the epithelial barrier integrity. Through bacterial adhesion assays and in vitro pull-down assays, we identified fibronectin-binding proteins (FnBP) of S. aureus as a key component that binds to CD47. Furthermore, we found that ciliated cell-specific CD47 deficiency or neutralizing antibody-mediated CD47 inactivation enhanced in vivo survival rates. These findings suggest that interfering with the interaction between airway epithelial CD47 and pathogenic bacterial FnBP holds promise for alleviating the adverse effects of super-infection.

Skin microbe-dependent TSLP-ILC2 priming axis in early life is co-opted in allergic inflammation.

Although early life colonization of commensal microbes contributes to long-lasting immune imprinting in host tissues, little is known regarding the pathophysiological consequences of postnatal microbial tuning of cutaneous immunity. Here, we show that postnatal exposure to specific skin commensal Staphylococcus lentus (S. lentus) promotes the extent of atopic dermatitis (AD)-like inflammation in adults through priming of group 2 innate lymphoid cells (ILC2s). Early postnatal skin is dynamically populated by discrete subset of primed ILC2s driven by microbiota-dependent induction of thymic stromal lymphopoietin (TSLP) in keratinocytes. Specifically, the indole-3-aldehyde-producing tryptophan metabolic pathway, shared across Staphylococcus species, is involved in TSLP-mediated ILC2 priming. Furthermore, we demonstrate a critical contribution of the early postnatal S. lentus-TSLP-ILC2 priming axis in facilitating AD-like inflammation that is not replicated by later microbial exposure. Thus, our findings highlight the fundamental role of time-dependent neonatal microbial-skin crosstalk in shaping the threshold of innate type 2 immunity co-opted in adulthood.

NADPH Oxidase 4-mediated Alveolar Macrophage Recruitment to Lung Attenuates Neutrophilic Inflammation in Staphylococcus aureus Infection.

When the lungs are infected with bacteria, alveolar macrophages (AMs) are recruited to the site and play a crucial role in protecting the host by reducing excessive lung inflammation. However, the regulatory mechanisms that trigger the recruitment of AMs to lung alveoli during an infection are still not fully understood. In this study, we identified a critical role for NADPH oxidase 4 (NOX4) in the recruitment of AMs during Staphylococcus aureus lung infection. We found that NOX4 knockout (KO) mice showed decreased recruitment of AMs and increased lung neutrophils and injury in response to S. aureus infection compared to wild-type (WT) mice. Interestingly, the burden of S. aureus in the lungs was not different between NOX4 KO and WT mice. Furthermore, we observed that depletion of AMs in WT mice during S. aureus infection increased the number of neutrophils and lung injury to a similar level as that observed in NOX4 KO mice. Additionally, we found that expression of intercellular adhesion molecule-1 (ICAM1) in NOX4 KO mice-derived lung endothelial cells was lower than that in WT mice-derived endothelial cells. Therefore, we conclude that NOX4 plays a crucial role in inducing the recruitment of AMs by controlling ICAM1 expression in lung endothelial cells, which is responsible for resolving lung inflammation during acute S. aureus infection.

Secreted Akkermansia muciniphila threonyl-tRNA synthetase functions to monitor and modulate immune homeostasis.

Commensal bacteria are critically involved in the establishment of tolerance against inflammatory challenges, the molecular mechanisms of which are just being uncovered. All kingdoms of life produce aminoacyl-tRNA synthetases (ARSs). Thus far, the non-translational roles of ARSs have largely been reported in eukaryotes. Here, we report that the threonyl-tRNA synthetase (AmTARS) of the gut-associated bacterium Akkermansia muciniphila is secreted and functions to monitor and modulate immune homeostasis. Secreted AmTARS triggers M2 macrophage polarization and orchestrates the production of anti-inflammatory IL-10 via its unique, evolutionary-acquired regions, which mediates specific interactions with TLR2. This interaction activates the MAPK and PI3K/AKT signaling pathways, which converge on CREB, leading to an efficient production of IL-10 and suppression of the central inflammatory mediator NF-κB. AmTARS restores IL-10-positive macrophages, increases IL-10 levels in the serum, and attenuates the pathological effects in colitis mice. Thus, commensal tRNA synthetases can act as intrinsic mediators that maintain homeostasis.

Amelioration of SARS-CoV-2 infection by ANO6 phospholipid scramblase inhibition.

As an enveloped virus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) delivers its viral genome into host cells via fusion of the viral and cell membranes. Here, we show that ANO6/TMEM16F-mediated cell surface exposure of phosphatidylserine is critical for SARS-CoV-2 entry and that ANO6-selective inhibitors are effective against SARS-CoV-2 infections. Application of the SARS-CoV-2 Spike pseudotyped virus (SARS2-PsV) evokes a cytosolic Ca2+ elevation and ANO6-dependent phosphatidylserine externalization in ACE2/TMPRSS2-positive mammalian cells. A high-throughput screening of drug-like chemical libraries identifies three different structural classes of chemicals showing ANO6 inhibitory effects. Among them, A6-001 displays the highest potency and ANO6 selectivity and it inhibits the single-round infection of SARS2-PsV in ACE2/TMPRSS2-positive HEK 293T cells. More importantly, A6-001 strongly inhibits authentic SARS-CoV-2-induced phosphatidylserine scrambling and SARS-CoV-2 viral replications in Vero, Calu-3, and primarily cultured human nasal epithelial cells. These results provide mechanistic insights into the viral entry process and offer a potential target for pharmacological intervention to protect against coronavirus disease 2019 (COVID-19).

Impairment of Mitochondrial ATP Synthesis Induces RIPK3-dependent Necroptosis in Lung Epithelial Cells During Lung Injury by Lung Inflammation.

Dysfunction of mitochondrial metabolism is implicated in cellular injury and cell death. While mitochondrial dysfunction is associated with lung injury by lung inflammation, the mechanism by which the impairment of mitochondrial ATP synthesis regulates necroptosis during acute lung injury (ALI) by lung inflammation is unclear. Here, we showed that the impairment of mitochondrial ATP synthesis induces receptor interacting serine/threonine kinase 3 (RIPK3)-dependent necroptosis during lung injury by lung inflammation. We found that the impairment of mitochondrial ATP synthesis by oligomycin, an inhibitor of ATP synthase, resulted in increased lung injury and RIPK3 levels in lung tissues during lung inflammation by LPS in mice. The elevated RIPK3 and RIPK3 phosphorylation levels by oligomycin resulted in high mixed lineage kinase domain-like (MLKL) phosphorylation, the terminal molecule in necroptotic cell death pathway, in lung epithelial cells during lung inflammation. Moreover, the levels of protein in bronchoalveolar lavage fluid (BALF) were increased by the activation of necroptosis via oligomycin during lung inflammation. Furthermore, the levels of ATP5A, a catalytic subunit of the mitochondrial ATP synthase complex for ATP synthesis, were reduced in lung epithelial cells of lung tissues from patients with acute respiratory distress syndrome (ARDS), the most severe form of ALI. The levels of RIPK3, RIPK3 phosphorylation and MLKL phosphorylation were elevated in lung epithelial cells in patients with ARDS. Our results suggest that the impairment of mitochondrial ATP synthesis induces RIPK3-dependent necroptosis in lung epithelial cells during lung injury by lung inflammation.

Proteomic analysis-based discovery of a novel biomarker that differentiates intestinal Behçet's disease from Crohn's disease.

Intestinal Behçet's disease (BD) and Crohn's disease (CD) present similar manifestations, but there are no specific diagnostic tests to differentiate them. We used a proteomic approach to discover novel diagnostic biomarkers specific to intestinal BD. Colon mucosa tissue samples were obtained from patients with intestinal BD or CD using colonoscopy-guided biopsy of the affected bowel. Peptides from seven intestinal BD and seven CD patients were extracted and labeled using tandem mass tag (TMT) reagents. The labeled peptides were identified and quantified using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The proteins were further validated using immunohistochemical (IHC) analysis with tissue samples and an ELISA test with serum samples from 20 intestinal BD and 20 CD patients. Using TMT/LC-MS/MS-based proteomic quantification, we identified 39 proteins differentially expressed between intestinal BD and CD. Beta-2 glycoprotein 1 (APOH) and maltase-glucoamylase (MGAM) showed higher intensity in the IHC staining of intestinal BD tissues than in CD tissues. The serum MGAM level was higher in intestinal BD patients. Proteomic analysis revealed that some proteins were differentially expressed in patients with intestinal BD compared with those with CD. Differential MGAM expression in intestinal BD suggests its role as a potential novel diagnostic biomarker.

Inhibition of Pendrin by a small molecule reduces Lipopolysaccharide-induced acute Lung Injury.

Rationale: Pendrin is encoded by SLC26A4 and its mutation leads to congenital hearing loss. Additionally, pendrin is up-regulated in inflammatory airway diseases such as chronic obstructive pulmonary disease, allergic rhinitis, and asthma. In this study, the effects of a novel pendrin inhibitor, YS-01, were investigated in an LPS-induced acute lung injury (ALI) mice model, and the mechanism underlying the effect of YS-01 was examined. Methods: Lipopolysaccharide (LPS, 10 mg/kg) was intranasally instilled in wild type (WT) and pendrin-null mice. YS-01 (10 mg/kg) was administered intra-peritoneally before or after LPS inhalation. Lung injury parameters were assessed in the lung tissue and bronchoalveolar lavage fluid (BALF). Pendrin levels in the BALF of 41 patients with acute respiratory distress syndrome (ARDS) due to pneumonia and 25 control (solitary pulmonary nodule) patients were also measured. Results: LPS instillation induced lung injury in WT mice but not in pendrin-null mice. Pendrin expression was increased by LPS stimulation both in vitro and in vivo. YS-01 treatment dramatically attenuated lung injury and reduced BALF cell counts and protein concentration after LPS instillation in WT mice. Proinflammatory cytokines and NF-κB activation were suppressed by YS-01 treatment in LPS-induced ALI mice. In BALF of patients whose ARDS was caused by pneumonia, pendrin expression was up-regulated compared to that in controls (mean, 24.86 vs. 6.83 ng/mL, P < 0.001). Conclusions: A novel pendrin inhibitor, YS-01, suppressed lung injury in LPS-induced ALI mice and our data provide a new strategy for the treatment of inflammatory airway diseases including sepsis-induced ALI.

Quantitative Proteomic Analysis of the Expression of SARS-CoV-2 Receptors in the Gut of Patients with Chronic Enterocolitis.

The cellular entry of severe respiratory syndrome coronavirus-2 (SARS-CoV-2) is mediated by interaction with the human angiotensin-converting enzyme 2 (ACE2), a receptor that is expressed on both lung and intestinal epithelial cells. We performed a quantitative proteomic analysis to investigate the expression of possible receptors for SARS-CoV-2 in the intestinal mucosa of 23 patients with chronic colitis. ACE2 expression was low and remained unaltered in the gut of patients with ulcerative colitis (UC), Crohn's disease (CD), intestinal Behcet's disease (BD), and intestinal tuberculosis (TB), when compared with that of healthy individuals. Additionally, the expression levels of some probable co-receptors, including dipeptidyl peptidase 4 (DPP4), aminopeptidase N (AMPN), and glutamyl aminopeptidase (AMPE), were unchanged in the affected UC, CD, intestinal BD, and intestinal TB colon mucosa samples. In conclusion, gut inflammation associated with chronic colitis does not mediate a further increase in the cellular entry of SARS-CoV-2.

NOX4 Deficiency Exacerbates the Impairment of Cystatin C-Dependent Hippocampal Neurogenesis by a Chronic High Fat Diet.

Hippocampal neurogenesis is linked with a cognitive process under a normal physiological condition including learning, memory, pattern separation, and cognitive flexibility. Hippocampal neurogenesis is altered by multiple factors such as the systemic metabolic changes. NADPH oxidase 4 (NOX4) has been implicated in the regulation of brain function. While the role of NOX4 plays in the brain, the mechanism by which NOX4 regulates hippocampal neurogenesis under metabolic stress is unclear. In this case, we show that NOX4 deficiency exacerbates the impairment of hippocampal neurogenesis by inhibiting neuronal maturation by a chronic high fat diet (HFD). NOX4 deficiency resulted in less hippocampal neurogenesis by decreasing doublecortin (DCX)-positive neuroblasts, a neuronal differentiation marker, and their branched-dendrites. Notably, NOX4 deficiency exacerbates the impairment of hippocampal neurogenesis by chronic HFD. Moreover, NOX4 deficiency had a significant reduction of Cystatin C levels, which is critical for hippocampal neurogenesis, under chronic HFD as well as normal chow (NC) diet. Furthermore, the reduction of Cystatin C levels was correlated with the impairment of hippocampal neurogenesis in NOX4 deficient and wild-type (WT) mice under chronic HFD. Our results suggest that NOX4 regulates the impairment of Cystatin C-dependent hippocampal neurogenesis under chronic HFD.

ADCK4 Deficiency Destabilizes the Coenzyme Q Complex, Which Is Rescued by 2,4-Dihydroxybenzoic Acid Treatment.

BACKGROUND: Mutations in ADCK4 (aarF domain containing kinase 4) generally manifest as steroid-resistant nephrotic syndrome and induce coenzyme Q10 (CoQ10) deficiency. However, the molecular mechanisms underlying steroid-resistant nephrotic syndrome resulting from ADCK4 mutations are not well understood, largely because the function of ADCK4 remains unknown. METHODS: To elucidate the ADCK4's function in podocytes, we generated a podocyte-specific, Adck4-knockout mouse model and a human podocyte cell line featuring knockout of ADCK4. These knockout mice and podocytes were then treated with 2,4-dihydroxybenzoic acid (2,4-diHB), a CoQ10 precursor analogue, or with a vehicle only. We also performed proteomic mass spectrometry analysis to further elucidate ADCK4's function. RESULTS: Absence of Adck4 in mouse podocytes caused FSGS and albuminuria, recapitulating features of nephrotic syndrome caused by ADCK4 mutations. In vitro studies revealed that ADCK4-knockout podocytes had significantly reduced CoQ10 concentration, respiratory chain activity, and mitochondrial potential, and subsequently displayed an increase in the number of dysmorphic mitochondria. However, treatment of 3-month-old knockout mice or ADCK4-knockout cells with 2,4-diHB prevented the development of renal dysfunction and reversed mitochondrial dysfunction in podocytes. Moreover, ADCK4 interacted with mitochondrial proteins such as COQ5, as well as cytoplasmic proteins such as myosin and heat shock proteins. Thus, ADCK4 knockout decreased the COQ complex level, but overexpression of ADCK4 in ADCK4-knockout podocytes transfected with wild-type ADCK4 rescued the COQ5 level. CONCLUSIONS: Our study shows that ADCK4 is required for CoQ10 biosynthesis and mitochondrial function in podocytes, and suggests that ADCK4 in podocytes stabilizes proteins in complex Q in podocytes. Our study also suggests a potential treatment strategy for nephrotic syndrome resulting from ADCK4 mutations.

Functional dynamics of bacterial species in the mouse gut microbiome revealed by metagenomic and metatranscriptomic analyses.

BACKGROUND: Microbial communities of the mouse gut have been extensively studied; however, their functional roles and regulation are yet to be elucidated. Metagenomic and metatranscriptomic analyses may allow us a comprehensive profiling of bacterial composition and functions of the complex gut microbiota. The present study aimed to investigate the active functions of the microbial communities in the murine cecum by analyzing both metagenomic and metatranscriptomic data on specific bacterial species within the microbial communities, in addition to the whole microbiome. RESULTS: Bacterial composition of the healthy mouse gut microbiome was profiled using the following three different approaches: 16S rRNA-based profiling based on amplicon and shotgun sequencing data, and genome-based profiling based on shotgun sequencing data. Consistently, Bacteroidetes, Firmicutes, and Deferribacteres emerged as the major phyla. Based on NCBI taxonomy, Muribaculaceae, Lachnospiraceae, and Deferribacteraceae were the predominant families identified in each phylum. The genes for carbohydrate metabolism were upregulated in Muribaculaceae, while genes for cofactors and vitamin metabolism and amino acid metabolism were upregulated in Deferribacteraceae. The genes for translation were commonly enhanced in all three families. Notably, combined analysis of metagenomic and metatranscriptomic sequencing data revealed that the functions of translation and metabolism were largely upregulated in all three families in the mouse gut environment. The ratio of the genes in the metagenome and their expression in the metatranscriptome indicated higher expression of carbohydrate metabolism in Muribaculum, Duncaniella, and Mucispirillum. CONCLUSIONS: We demonstrated a fundamental methodology for linking genomic and transcriptomic datasets to examine functional activities of specific bacterial species in a complicated microbial environment. We investigated the normal flora of the mouse gut using three different approaches and identified Muribaculaceae, Lachnospiraceae, and Deferribacteraceae as the predominant families. The functional distribution of these families was reflected in the entire microbiome. By comparing the metagenomic and metatranscriptomic data, we found that the expression rates differed for different functional categories in the mouse gut environment. Application of these methods to track microbial transcription in individuals over time, or before and after administration of a specific stimulus will significantly facilitate future development of diagnostics and treatments.

Apolipoprotein E and Periostin Are Potential Biomarkers of Nasal Mucosal Inflammation. A Parallel Approach of In Vitro and In Vivo Secretomes.

No previously suggested biomarkers of nasal mucosal inflammation have been practically applied in clinical fields, and nasal epithelium-derived secreted proteins as biomarkers have not specifically been investigated. The goal of this study was to identify secreted proteins that dynamically change during the differentiation from basal cells to fully differentiated cells and examine whether nasal epithelium-derived proteins can be used as biomarkers of nasal mucosal inflammation, such as chronic rhinosinusitis. To achieve this goal, we analyzed two secretomes using the isobaric tag for relative and absolute quantification technique. From in vitro secretomes, we identified the proteins altered in apical secretions of primary human nasal epithelial cells according to the degree of differentiation; from in vivo secretomes, we identified the increased proteins in nasal lavage fluids obtained from patients 2 weeks after endoscopic sinus surgery for chronic sinusitis. We then used a parallel approach to identify specific biomarkers of nasal mucosal inflammation; first, we selected apolipoprotein E as a nasal epithelial cell-derived biomarker through screening proteins that were upregulated in both in vitro and in vivo secretomes, and verified highly secreted apolipoprotein E in nasal lavage fluids of the patients by Western blotting. Next, we selected periostin as an inflammatory mediator-inducible biomarker from in vivo secretomes, the secretion of which was not induced under in vitro culture conditions. We demonstrated that those two nasal epithelium-derived proteins are possible biomarkers of nasal mucosal inflammation.

Commensal-derived metabolites govern Vibrio cholerae pathogenesis in host intestine.

BACKGROUND: Recent evidence suggests that the commensal microbes act as a barrier against invading pathogens and enteric infections are the consequences of multi-layered interactions among commensals, pathogens, and the host intestinal tissue. However, it remains unclear how perturbations of the gut microbiota compromise host infection resistance, especially through changes at species and metabolite levels. RESULTS: Here, we illustrate how Bacteroides vulgatus, a dominant species of the Bacteroidetes phylum in mouse intestine, suppresses infection by Vibrio cholerae, an important human pathogen. Clindamycin (CL) is an antibiotic that selectively kills anaerobic bacteria, and accordingly Bacteroidetes are completely eradicated from CL-treated mouse intestines. The Bacteroidetes-depleted adult mice developed severe cholera-like symptoms, when infected with V. cholerae. Germ-free mice mono-associated with B. vulgatus became resistant to V. cholerae infection. Levels of V. cholerae growth-inhibitory metabolites including short-chain fatty acids plummeted upon CL treatment, while levels of compounds that enhance V. cholerae proliferation were elevated. Furthermore, the intestinal colonization process of V. cholerae was well-simulated in CL-treated adult mice. CONCLUSIONS: Overall, we provide insights into how a symbiotic microbe and a pathogenic intruder interact inside host intestine. We identified B. vulgatus as an indigenous microbial species that can suppress intestinal infection. Our results also demonstrate that commensal-derived metabolites are a critical determinant for host resistance against V. cholerae infection, and that CL pretreatment of adult mice generates a simple yet useful model of cholera infection.

Naphthalene-based fluorescent probes for glutathione and their applications in living cells and patients with sepsis.

Rationale: Among the biothiols-related diseases, sepsis is defined as life-threatening organ dysfunction caused by a dysregulated host response to infection and can result in severe oxidative stress and damage to multiple organs. In this study, we aimed to develop a fluorescence chemosensor that can both detect GSH and further predict sepsis. Methods: In this study, two new naphthalene dialdehyde compounds containing different functional groups were synthesized, and the sensing abilities of these compounds towards biothiols and its applications for prediction of sepsis were investigated. Results: Our study revealed that the newly developed probe 6-methoxynaphthalene-2, 3-dicarbaldehyde (MNDA) has two-photon is capable of detecting GSH in live cells with two-photon microscopy (TPM) under the excitation at a wavelength of 900 nm. Furthermore, two GSH detection probes naphthalene-2,3-dicarboxaldehyde (NDA) and 6-fluoronaphthalene-2,3-dicarbaldehyde (FNDA) not only can detect GSH in living cells, but also showed clinical significance for the diagnosis and prediction of mortality in patients with sepsis. Conclusions: These results open up a promising direction for further medical diagnostic techniques.

A Far-Red-Emitting Fluorescence Probe for Sensitive and Selective Detection of Peroxynitrite in Live Cells and Tissues.

In this study, the far-red-emitting fluorescence probe 1, containing a rhodamine derivative and a hydrazide reactive group, was developed for peroxynitrite detection and imaging. This probe, which is cell permeable and shows high sensitivity and selectivity in fluorometric detection of peroxynitrite over other ROS/RNS, was successfully utilized to detect exogenous and endogenous peroxynitrite in HeLa and RAW 264.7 cells, respectively. More importantly, 1 can also be used to detect endogenous peroxynitrite generated in Pseudomonas aeruginosa (PAO1)-infected mouse bone marrow-derived neutrophils. We anticipate that the new probe will serve as a powerful molecular imaging tool in investigations of the role(s) played by peroxynitrite in a variety of physiological and pathological contexts.

Mitophagy: a balance regulator of NLRP3 inflammasome activation.

The NLRP3 inflammasome is activated by a variety of external or host-derived stimuli and its activation initiates an inflammatory response through caspase-1 activation, resulting in inflammatory cytokine IL-1ß maturation and secretion. The NLRP3 inflammasome activation is a kind of innate immune response, most likely mediated by myeloid cells acting as a host defense mechanism. However, if this activation is not properly regulated, excessive inflammation induced by overactivated NLRP3 inflammasome can be detrimental to the host, causing tissue damage and organ dysfunction, eventually causing several diseases. Previous studies have suggested that mitochondrial damage may be a cause of NLRP3 inflammasome activation and autophagy, which is a conserved self-degradation process that negatively regulates NLRP3 inflammasome activation. Recently, mitochondria-selective autophagy, termed mitophagy, has emerged as a central player for maintaining mitochondrial homeostasis through the elimination of damaged mitochondria, leading to the prevention of hyperinflammation triggered by NLRP3 inflammasome activation. In this review, we will first focus on the molecular mechanisms of NLRP3 inflammasome activation and NLRP3 inflammasome-related diseases. We will then discuss autophagy, especially mitophagy, as a negative regulator of NLPP3 inflammasome activation by examining recent advances in research. [BMB Reports 2016; 49(10): 529-535].

Synthesis of a highly HOCl-selective fluorescent probe and its use for imaging HOCl in cells and organisms.

During infection, nicotinamide adenine dinucleotide phosphate-oxidase of innate immune cells generates important microbicidal reactive oxygen species (ROS) such as hypochlorous acid (HOCl) to kill the invading pathogens. However, excess amounts of HOCl induce oxidative damage of functional biomolecules such as DNA and proteins, which may cause chronic inflammatory diseases. Herein, we outline protocols for the preparation of a rhodamine-based HOCl probe, as well as applications thereof, with which to detect HOCl in living cells and organisms. The probe (R19S) can be prepared from a commercially available rhodamine, rhodamine 6G, in two steps. When R19S is treated with HOCl, the sulfur atom is replaced by an oxygen atom, resulting in opening of the lactone ring; thus, nonfluorescent R19S is converted to highly fluorescent rhodamine 19 (R19). R19S exhibits high selectivity for HOCl over other ROS and high sensitivity in a weakly acidic environment. In addition, we describe fluorescence imaging assays of HOCl in mouse neutrophils and Drosophila targeted using this probe. The approximate amount of time required to synthesize the probe is 2-3 d, after which it can be used for up to 5 h in the bioimaging of living cells.

SESN2/sestrin2 suppresses sepsis by inducing mitophagy and inhibiting NLRP3 activation in macrophages.

Proper regulation of mitophagy for mitochondrial homeostasis is important in various inflammatory diseases. However, the precise mechanisms by which mitophagy is activated to regulate inflammatory responses remain largely unknown. The NLRP3 (NLR family, pyrin domain containing 3) inflammasome serves as a platform that triggers the activation of CASP1 (caspase 1) and secretion of proinflammatory cytokines. Here, we demonstrate that SESN2 (sestrin 2), known as stress-inducible protein, suppresses prolonged NLRP3 inflammasome activation by clearance of damaged mitochondria through inducing mitophagy in macrophages. SESN2 plays a dual role in inducing mitophagy in response to inflammasome activation. First, SESN2 induces "mitochondrial priming" by marking mitochondria for recognition by the autophagic machinery. For mitochondrial preparing, SESN2 facilitates the perinuclear-clustering of mitochondria by mediating aggregation of SQSTM1 (sequestosome 1) and its binding to lysine 63 (Lys63)-linked ubiquitins on the mitochondrial surface. Second, SESN2 activates the specific autophagic machinery for degradation of primed mitochondria via an increase of ULK1 (unc-51 like kinase 1) protein levels. Moreover, increased SESN2 expression by extended LPS (lipopolysaccharide) stimulation is mediated by NOS2 (nitric oxide synthase 2, inducible)-mediated NO (nitric oxide) in macrophages. Thus, Sesn2-deficient mice displayed defective mitophagy, which resulted in hyperactivation of inflammasomes and increased mortality in 2 different sepsis models. Our findings define a unique regulatory mechanism of mitophagy activation for immunological homeostasis that protects the host from sepsis.