The tetraspanin CD53 protects stressed hematopoietic stem cells via promotion of DREAM complex-mediated quiescence.

The hematopoietic stem cell (HSC) cycle responds to inflammatory and other proliferative stressors; however, these cells must quickly return to quiescence to avoid exhaustion and maintain their functional integrity. The mechanisms that regulate this return to quiescence are not well understood. Here, we show that tetraspanin CD53 is markedly upregulated in HSCs in response to a variety of inflammatory and proliferative stimuli and that the loss of CD53 is associated with prolonged cycling and reduced HSC function in the context of inflammatory stress. Mechanistically, CD53 promotes the activity of the dimerization partner, RB-like, E2F, and multi-vulva class B (DREAM) transcriptional repressor complex, which downregulates genes associated with cycling and division. Proximity labeling and confocal fluorescence microscopy studies showed that CD53 interacts with DREAM-associated proteins, specifically promoting the interaction between Rbl2/p130 and its phosphatase protein phosphatase 2A (PP2A), effectively stabilizing p130 protein availability for DREAM binding. Together, these data identified a novel mechanism by which stressed HSCs resist cycling.

Preclinical mouse model of a misfolded PNLIP variant develops chronic pancreatitis.

OBJECTIVE: Increasing evidence implicates mutation-induced protein misfolding and endoplasm reticulum (ER) stress in the pathophysiology of chronic pancreatitis (CP). The paucity of animal models harbouring genetic risk variants has hampered our understanding of how misfolded proteins trigger CP. We previously showed that pancreatic triglyceride lipase (PNLIP) p.T221M, a variant associated with steatorrhoea and possibly CP in humans, misfolds and elicits ER stress in vitro suggesting proteotoxicity as a potential disease mechanism. Our objective was to create a mouse model to determine if PNLIP p.T221M causes CP and to define the mechanism. DESIGN: We created a mouse model of Pnlip p.T221M and characterised the structural and biochemical changes in the pancreas aged 1-12 months. We used multiple methods including histochemistry, immunostaining, transmission electron microscopy, biochemical assays, immunoblotting and qPCR. RESULTS: We demonstrated the hallmarks of human CP in Pnlip p.T221M homozygous mice including progressive pancreatic atrophy, acinar cell loss, fibrosis, fatty change, immune cell infiltration and reduced exocrine function. Heterozygotes also developed CP although at a slower rate. Immunoblot showed that pancreatic PNLIP T221M misfolded as insoluble aggregates. The level of aggregates in homozygotes declined with age and was much lower in heterozygotes at all ages. The Pnlip p.T221M pancreas had increased ER stress evidenced by dilated ER, increased Hspa5 (BiP) mRNA abundance and a maladaptive unfolded protein response leading to upregulation of Ddit3 (CHOP), nuclear factor-κB and cell death. CONCLUSION: Expression of PNLIP p.T221M in a preclinical mouse model results in CP caused by ER stress and proteotoxicity of misfolded mutant PNLIP.

NADPH oxidase controls pulmonary neutrophil infiltration in the response to fungal cell walls by limiting LTB4.

Leukocyte reduced NADP (NADPH) oxidase plays a key role in host defense and immune regulation. Genetic defects in NADPH oxidase result in chronic granulomatous disease (CGD), characterized by recurrent bacterial and fungal infections and aberrant inflammation. Key drivers of hyperinflammation induced by fungal cell walls in CGD are still incompletely defined. In this study, we found that CGD (CYBB-) neutrophils produced higher amounts of leukotriene B4 (LTB4) in vitro after activation with zymosan or immune complexes, compared with wild-type (WT) neutrophils. This finding correlated with increased calcium influx in CGD neutrophils, which was restrained in WT neutrophils by the electrogenic activity of NADPH oxidase. Increased LTB4 generation by CGD neutrophils was also augmented by paracrine cross talk with the LTB4 receptor BLT1. CGD neutrophils formed more numerous and larger clusters in the presence of zymosan in vitro compared with WT cells, and the effect was also LTB4- and BLT1-dependent. In zymosan-induced lung inflammation, focal neutrophil infiltrates were increased in CGD compared with WT mice and associated with higher LTB4 levels. Inhibiting LTB4 synthesis or antagonizing the BLT1 receptor after zymosan challenge reduced lung neutrophil recruitment in CGD to WT levels. Thus, LTB4 was the major driver of excessive neutrophilic lung inflammation in CGD mice in the early response to fungal cell walls, likely by a dysregulated feed-forward loop involving amplified neutrophil production of LTB4. This study identifies neutrophil LTB4 generation as a target of NADPH oxidase regulation, which could potentially be exploited therapeutically to reduce excessive inflammation in CGD.

Autophagy genes in myeloid cells counteract IFNγ-induced TNF-mediated cell death and fatal TNF-induced shock.

Host inflammatory responses must be tightly regulated to ensure effective immunity while limiting tissue injury. IFN gamma (IFNγ) primes macrophages to mount robust inflammatory responses. However, IFNγ also induces cell death, and the pathways that regulate IFNγ-induced cell death are incompletely understood. Using genome-wide CRISPR/Cas9 screening, we identified autophagy genes as central mediators of myeloid cell survival during the IFNγ response. Hypersensitivity of autophagy gene-deficient cells to IFNγ was mediated by tumor necrosis factor (TNF) signaling via receptor interacting protein kinase 1 (RIPK1)- and caspase 8-mediated cell death. Mice with myeloid cell-specific autophagy gene deficiency exhibited marked hypersensitivity to fatal systemic TNF administration. This increased mortality in myeloid autophagy gene-deficient mice required the IFNγ receptor, and mortality was completely reversed by pharmacologic inhibition of RIPK1 kinase activity. These findings provide insight into the mechanism of IFNγ-induced cell death via TNF, demonstrate a critical function of autophagy genes in promoting cell viability in the presence of inflammatory cytokines, and implicate this cell survival function in protection against mortality during the systemic inflammatory response.

Functional characterization of four ATP-binding cassette transporter A3 gene (ABCA3) variants.

ABCA3 transports phospholipids across lamellar body membranes in pulmonary alveolar type II cells and is required for surfactant assembly. Rare, biallelic, pathogenic ABCA3 variants result in lethal neonatal respiratory distress syndrome and childhood interstitial lung disease. Qualitative functional characterization of ABCA3 missense variants suggests two pathogenic classes: disrupted intracellular trafficking (type I mutant) or impaired ATPase-mediated phospholipid transport into the lamellar bodies (type II mutant). We qualitatively compared wild-type (WT-ABCA3) with four uncharacterized ABCA3 variants (c.418A>C;p.Asn140His, c.3609_3611delCTT;p.Phe1203del, c.3784A>G;p.Ser1262Gly, and c.4195G>A;p.Val1399Met) in A549 cells using protein processing, colocalization with intracellular organelles, lamellar body ultrastructure, and ATPase activity. We quantitatively measured lamellar body-like vesicle diameter and intracellular ABCA3 trafficking using fluorescence-based colocalization. Three ABCA3 variants (p.Asn140His, p.Ser1262Gly, and p.Val1399Met) were processed and trafficked normally and demonstrated well-organized lamellar body-like vesicles, but had reduced ATPase activity consistent with type II mutants. P.Phe1203del was processed normally, had reduced ATPase activity, and well-organized lamellar body-like vesicles, but quantitatively colocalized with both endoplasmic reticulum and lysosomal markers, an intermediate phenotype suggesting disruption of both intracellular trafficking and phospholipid transport. All ABCA3 mutants demonstrated mean vesicle diameters smaller than WT-ABCA3. Qualitative and quantitative functional characterization of ABCA3 variants informs mechanisms of pathogenicity.

Enteroviruses infect human enteroids and induce antiviral signaling in a cell lineage-specific manner.

Enteroviruses are among the most common viral infectious agents of humans and are primarily transmitted by the fecal-oral route. However, the events associated with enterovirus infections of the human gastrointestinal tract remain largely unknown. Here, we used stem cell-derived enteroids from human small intestines to study enterovirus infections of the intestinal epithelium. We found that enteroids were susceptible to infection by diverse enteroviruses, including echovirus 11 (E11), coxsackievirus B (CVB), and enterovirus 71 (EV71), and that contrary to an immortalized intestinal cell line, enteroids induced antiviral and inflammatory signaling pathways in response to infection in a virus-specific manner. Furthermore, using the Notch inhibitor dibenzazepine (DBZ) to drive cellular differentiation into secretory cell lineages, we show that although goblet cells resist E11 infection, enteroendocrine cells are permissive, suggesting that enteroviruses infect specific cell populations in the human intestine. Taken together, our studies provide insights into enterovirus infections of the human intestine, which could lead to the identification of novel therapeutic targets and/or strategies to prevent or treat infections by these highly clinically relevant viruses.

Serum squamous cell carcinoma antigen as an early indicator of response during therapy of cervical cancer.

BACKGROUND: Pretreatment serum squamous cell carcinoma antigen (SCCA) is a prognostic biomarker in women with cervical cancer. SCCA has not been evaluated as an early indicator of response to chemoradiation therapy (CRT). The molecular role of the two SCCA isoforms, SCCA1 (SERPINB3) and SCCA2 (SERPINB4), in cervical cancer is unknown. We hypothesised that changes in serum SCCA during definitive CRT predicts treatment response, and that SCCA1 mediates radiation resistance. METHODS: Patients treated with definitive CRT for cervical squamous carcinoma with serum SCCA measured were included. SCCA immunohistochemistry was performed on tumour biopsies. Post-treatment FDG-PET/CT, recurrence, and overall survival were recorded. Radiation response of cervical tumour cell lines after SCCA1 expression or CRISPR/Cas9 knockout was evaluated by clonogenic survival assay. RESULTS: Persistently elevated serum SCCA during definitive CRT was an independent predictor of positive post-therapy FDG-PET/CT (P=0.043), recurrence (P=0.0046) and death (P=0.015). An SCCA1-expressing vector increased radioresistance, while SCCA knock out increased radiosensitivity of cervical tumour cell lines in vitro. CONCLUSIONS: Early response assessment with serum SCCA is a powerful prognostic tool. These findings suggest that escalation of therapy in patients with elevated or sustained serum SCCA and molecular targeting of SCCA1 should be considered.

Activation of the Caenorhabditis elegans Degenerin Channel by Shear Stress Requires the MEC-10 Subunit.

Mechanotransduction in Caenorhabditis elegans touch receptor neurons is mediated by an ion channel formed by MEC-4, MEC-10, and accessory proteins. To define the role of these subunits in the channel's response to mechanical force, we expressed degenerin channels comprising MEC-4 and MEC-10 in Xenopus oocytes and examined their response to laminar shear stress (LSS). Shear stress evoked a rapid increase in whole cell currents in oocytes expressing degenerin channels as well as channels with a MEC-4 degenerin mutation (MEC-4d), suggesting that C. elegans degenerin channels are sensitive to LSS. MEC-10 is required for a robust LSS response as the response was largely blunted in oocytes expressing homomeric MEC-4 or MEC-4d channels. We examined a series of MEC-10/MEC-4 chimeras to identify specific domains (amino terminus, first transmembrane domain, and extracellular domain) and sites (residues 130-132 and 134-137) within MEC-10 that are required for a robust response to shear stress. In addition, the LSS response was largely abolished by MEC-10 mutations encoded by a touch-insensitive mec-10 allele, providing a correlation between the channel's responses to two different mechanical forces. Our findings suggest that MEC-10 has an important role in the channel's response to mechanical forces.

A genome-wide RNAi screen identifies potential drug targets in a C. elegans model of α1-antitrypsin deficiency.

α1-Antitrypsin deficiency (ATD) is a common genetic disorder that can lead to end-stage liver and lung disease. Although liver transplantation remains the only therapy currently available, manipulation of the proteostasis network (PN) by small molecule therapeutics offers great promise. To accelerate the drug-discovery process for this disease, we first developed a semi-automated high-throughput/content-genome-wide RNAi screen to identify PN modifiers affecting the accumulation of the α1-antitrypsin Z mutant (ATZ) in a Caenorhabditis elegans model of ATD. We identified 104 PN modifiers, and these genes were used in a computational strategy to identify human ortholog-ligand pairs. Based on rigorous selection criteria, we identified four FDA-approved drugs directed against four different PN targets that decreased the accumulation of ATZ in C. elegans. We also tested one of the compounds in a mammalian cell line with similar results. This methodology also proved useful in confirming drug targets in vivo, and predicting the success of combination therapy. We propose that small animal models of genetic disorders combined with genome-wide RNAi screening and computational methods can be used to rapidly, economically and strategically prime the preclinical discovery pipeline for rare and neglected diseases with limited therapeutic options.

A C. elegans model of human α1-antitrypsin deficiency links components of the RNAi pathway to misfolded protein turnover.

The accumulation of serpin oligomers and polymers within the endoplasmic reticulum (ER) causes cellular injury in patients with the classical form α1-antitrypsin deficiency (ATD). To better understand the cellular and molecular genetic aspects of this disorder, we generated transgenic C. elegans strains expressing either the wild-type (ATM) or Z mutant form (ATZ) of the human serpin fused to GFP. Animals secreted ATM, but retained polymerized ATZ within dilated ER cisternae. These latter animals also showed slow growth, smaller brood sizes and decreased longevity; phenotypes observed in ATD patients or transgenic mouse lines expressing ATZ. Similar to mammalian models, ATZ was disposed of by autophagy and ER-associated degradation pathways. Mutant strains defective in insulin signaling (daf-2) also showed a marked decrease in ATZ accumulation. Enhanced ATZ turnover was associated with the activity of two proteins central to systemic/exogenous (exo)-RNAi pathway: the dsRNA importer, SID-1 and the argonaute, RDE-1. Animals with enhanced exo-RNAi activity (rrf-3 mutant) phenocopied the insulin signaling mutants and also showed increased ATZ turnover. Taken together, these studies allude to the existence of a novel proteostasis pathway that mechanistically links misfolded protein turnover to components of the systemic RNAi machinery.

SERPINB12 Is a Slow-Binding Inhibitor of Granzyme A and Hepsin.

The clade B/intracellular serpins protect cells from peptidase-mediated injury by forming covalent complexes with their targets. SERPINB12 is expressed in most tissues, especially at cellular interfaces with the external environment. This wide tissue distribution pattern is similar to that of granzyme A (GZMA). Because SERPINB12 inhibits trypsin-like serine peptidases, we determined whether it might also neutralize GZMA. SERPINB12 formed a covalent complex with GZMA and inhibited the enzyme with typical serpin slow-binding kinetics. SERPINB12 also inhibited Hepsin. SERPINB12 may function as an endogenous inhibitor of these peptidases.

Non-microfluidic methods for imaging live C. elegans.

There are many challenges to live Caenorhabditis elegans imaging including the high motility of the animals and sustaining their viability for extended periods of time. Commonly used anesthetics to immobilize the C. elegans for imaging purpose prevents feeding of the animals and can cause cellular physiologic changes. Here we present three adapted or novel methodologies to image live C. elegans over different imaging microscopy equipment to allow for visualization of animals by DIC and fluorescence without the use of microfluidic technologies. The methods present here use common microscopy consumables and equipment found in many imaging core facilities and can be easily adapted to fit on multiple microscopy systems.

Isolation of serpin-interacting proteins in C. elegans using protein affinity purification.

Caenorhabditis elegans is a useful model organism for combining multiple imaging, genetic, and biochemical methodologies to gain more insight into the biological function of specific proteins. Combining both biochemical and genetic analyses can lead to a better understanding of how a given protein may function within the context of a network of other proteins or specific pathway. Here, we describe a protocol for the biochemical isolation of serpin-interacting proteins using affinity purification and proteomic analysis. As the knowledge of in vivo serpin interacting partners in C. elegans has largely been obtained using genetic and in vitro recombinant protein studies, this protocol serves as a complementary approach to provide insight into the biological function and regulation of serpins.

Microfluidic device facilitates in vitro modeling of human neonatal necrotizing enterocolitis-on-a-chip.

Necrotizing enterocolitis (NEC) is a deadly gastrointestinal disease of premature infants that is associated with an exaggerated inflammatory response, dysbiosis of the gut microbiome, decreased epithelial cell proliferation, and gut barrier disruption. We describe an in vitro model of the human neonatal small intestinal epithelium (Neonatal-Intestine-on-a-Chip) that mimics key features of intestinal physiology. This model utilizes intestinal enteroids grown from surgically harvested intestinal tissue from premature infants and cocultured with human intestinal microvascular endothelial cells within a microfluidic device. We used our Neonatal-Intestine-on-a-Chip to recapitulate NEC pathophysiology by adding infant-derived microbiota. This model, named NEC-on-a-Chip, simulates the predominant features of NEC, including significant upregulation of proinflammatory cytokines, decreased intestinal epithelial cell markers, reduced epithelial proliferation, and disrupted epithelial barrier integrity. NEC-on-a-Chip provides an improved preclinical model of NEC that facilitates comprehensive analysis of the pathophysiology of NEC using precious clinical samples. This model is an advance toward a personalized medicine approach to test new therapeutics for this devastating disease.

Staphylococcus aureus α-toxin impairs early neutrophil localization via electrogenic disruption of store-operated calcium entry.

The pore-forming S. aureus α-toxin (Hla) contributes to virulence and disease pathogenesis. While high concentrations of toxin induce cell death, neutrophils exhibit relative resistance to lysis, suggesting that the action of Hla may not be solely conferred by lytic susceptibility. Using intravital microscopy, we observed that Hla disrupts neutrophil localization and clustering early in infection. Hla forms a narrow, ion-selective pore, suggesting that Hla may dysregulate calcium or other ions to impair neutrophil function. We found that sub-lytic Hla did not permit calcium influx but caused rapid membrane depolarization. Depolarization decreases the electrogenic driving force for calcium, and concordantly, Hla suppressed calcium signaling in vitro and in vivo and calcium-dependent leukotriene B4 (LTB4) production, a key mediator of neutrophil clustering. Thus, Hla disrupts the early patterning of the neutrophil response to infection, in part through direct impairment of neutrophil calcium signaling. This early mis-localization of neutrophils may contribute to establishment of infection.

SERPINB3 (SCCA1) inhibits cathepsin L and lysoptosis, protecting cervical cancer cells from chemoradiation.

The endogenous lysosomal cysteine protease inhibitor SERPINB3 (squamous cell carcinoma antigen 1, SCCA1) is elevated in patients with cervical cancer and other malignancies. High serum SERPINB3 is prognostic for recurrence and death following chemoradiation therapy. Cervical cancer cells genetically lacking SERPINB3 are more sensitive to ionizing radiation (IR), suggesting this protease inhibitor plays a role in therapeutic response. Here we demonstrate that SERPINB3-deficient cells have enhanced sensitivity to IR-induced cell death. Knock out of SERPINB3 sensitizes cells to a greater extent than cisplatin, the current standard of care. IR in SERPINB3 deficient cervical carcinoma cells induces predominantly necrotic cell death, with biochemical and cellular features of lysoptosis. Rescue with wild-type SERPINB3 or a reactive site loop mutant indicates that protease inhibitory activity is required to protect cervical tumor cells from radiation-induced death. Transcriptomics analysis of primary cervix tumor samples and genetic knock out demonstrates a role for the lysosomal protease cathepsin L in radiation-induced cell death in SERPINB3 knock-out cells. These data support targeting of SERPINB3 and lysoptosis to treat radioresistant cervical cancers.

Lysoptosis is an evolutionarily conserved cell death pathway moderated by intracellular serpins.

Lysosomal membrane permeabilization (LMP) and cathepsin release typifies lysosome-dependent cell death (LDCD). However, LMP occurs in most regulated cell death programs suggesting LDCD is not an independent cell death pathway, but is conscripted to facilitate the final cellular demise by other cell death routines. Previously, we demonstrated that Caenorhabditis elegans (C. elegans) null for a cysteine protease inhibitor, srp-6, undergo a specific LDCD pathway characterized by LMP and cathepsin-dependent cytoplasmic proteolysis. We designated this cell death routine, lysoptosis, to distinguish it from other pathways employing LMP. In this study, mouse and human epithelial cells lacking srp-6 homologues, mSerpinb3a and SERPINB3, respectively, demonstrated a lysoptosis phenotype distinct from other cell death pathways. Like in C. elegans, this pathway depended on LMP and released cathepsins, predominantly cathepsin L. These studies suggested that lysoptosis is an evolutionarily-conserved eukaryotic LDCD that predominates in the absence of neutralizing endogenous inhibitors.

Serpins flex their muscle: II. Structural insights into target peptidase recognition, polymerization, and transport functions.

Inhibitory serpins are metastable proteins that undergo a substantial conformational rearrangement to covalently trap target peptidases. The serpin reactive center loop contributes a majority of the interactions that serpins make during the initial binding to target peptidases. However, structural studies on serpin-peptidase complexes reveal a broader set of contacts on the scaffold of inhibitory serpins that have substantial influence on guiding peptidase recognition. Structural and biophysical studies also reveal how aberrant serpin folding can lead to the formation of domain-swapped serpin multimers rather than the monomeric metastable state. Serpin domain swapping may therefore underlie the polymerization events characteristic of the serpinopathies. Finally, recent structural studies reveal how the serpin fold has been adapted for non-inhibitory functions such as hormone binding.

Serpins flex their muscle: I. Putting the clamps on proteolysis in diverse biological systems.

Serpins compose the largest superfamily of peptidase inhibitors and are well known as regulators of hemostasis and thrombolysis. Studies using model organisms, from plants to vertebrates, now show that serpins and their unique inhibitory mechanism and conformational flexibility are exploited to control proteolysis in molecular pathways associated with cell survival, development, and host defense. In addition, an increasing number of non-inhibitory serpins are emerging as important elements within a diversity of biological systems by serving as chaperones, hormone transporters, or anti-angiogenic factors.

Interleukin-22 signaling attenuates necrotizing enterocolitis by promoting epithelial cell regeneration.

Necrotizing enterocolitis (NEC) is a deadly intestinal inflammatory disorder that primarily affects premature infants and lacks adequate therapeutics. Interleukin (IL)-22 plays a critical role in gut barrier maintenance, promoting epithelial regeneration, and controlling intestinal inflammation in adult animal models. However, the importance of IL-22 signaling in neonates during NEC remains unknown. We investigated the role of IL-22 in the neonatal intestine under homeostatic and inflammatory conditions by using a mouse model of NEC. Our data reveal that Il22 expression in neonatal murine intestine is negligible until weaning, and both human and murine neonates lack IL-22 production during NEC. Mice deficient in IL-22 or lacking the IL-22 receptor in the intestine display a similar susceptibility to NEC, consistent with the lack of endogenous IL-22 during development. Strikingly, treatment with recombinant IL-22 during NEC substantially reduces inflammation and enhances epithelial regeneration. These findings may provide a new therapeutic strategy to attenuate NEC.