Publications by authors named "Bettina L Lee"

15 Publications

  • Page 1 of 1

NINJ1 mediates plasma membrane rupture during lytic cell death.

Nature 2021 Mar 20;591(7848):131-136. Epub 2021 Jan 20.

Department of Physiological Chemistry, Genentech Inc., South San Francisco, CA, USA.

Plasma membrane rupture (PMR) is the final cataclysmic event in lytic cell death. PMR releases intracellular molecules known as damage-associated molecular patterns (DAMPs) that propagate the inflammatory response. The underlying mechanism of PMR, however, is unknown. Here we show that the cell-surface NINJ1 protein, which contains two transmembrane regions, has an essential role in the induction of PMR. A forward-genetic screen of randomly mutagenized mice linked NINJ1 to PMR. Ninj1 macrophages exhibited impaired PMR in response to diverse inducers of pyroptotic, necrotic and apoptotic cell death, and were unable to release numerous intracellular proteins including HMGB1 (a known DAMP) and LDH (a standard measure of PMR). Ninj1 macrophages died, but with a distinctive and persistent ballooned morphology, attributable to defective disintegration of bubble-like herniations. Ninj1 mice were more susceptible than wild-type mice to infection with Citrobacter rodentium, which suggests a role for PMR in anti-bacterial host defence. Mechanistically, NINJ1 used an evolutionarily conserved extracellular domain for oligomerization and subsequent PMR. The discovery of NINJ1 as a mediator of PMR overturns the long-held idea that cell death-related PMR is a passive event.
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http://dx.doi.org/10.1038/s41586-021-03218-7DOI Listing
March 2021

MCC950/CRID3 potently targets the NACHT domain of wild-type NLRP3 but not disease-associated mutants for inflammasome inhibition.

PLoS Biol 2019 09 16;17(9):e3000354. Epub 2019 Sep 16.

Inflammation Research Center, VIB, Ghent, Belgium.

The nucleotide-binding-domain (NBD)-and leucine-rich repeat (LRR)-containing (NLR) family, pyrin-domain-containing 3 (NLRP3) inflammasome drives pathological inflammation in a suite of autoimmune, metabolic, malignant, and neurodegenerative diseases. Additionally, NLRP3 gain-of-function point mutations cause systemic periodic fever syndromes that are collectively known as cryopyrin-associated periodic syndrome (CAPS). There is significant interest in the discovery and development of diarylsulfonylurea Cytokine Release Inhibitory Drugs (CRIDs) such as MCC950/CRID3, a potent and selective inhibitor of the NLRP3 inflammasome pathway, for the treatment of CAPS and other diseases. However, drug discovery efforts have been constrained by the lack of insight into the molecular target and mechanism by which these CRIDs inhibit the NLRP3 inflammasome pathway. Here, we show that the NAIP, CIITA, HET-E, and TP1 (NACHT) domain of NLRP3 is the molecular target of diarylsulfonylurea inhibitors. Interestingly, we find photoaffinity labeling (PAL) of the NACHT domain requires an intact (d)ATP-binding pocket and is substantially reduced for most CAPS-associated NLRP3 mutants. In concordance with this finding, MCC950/CRID3 failed to inhibit NLRP3-driven inflammatory pathology in two mouse models of CAPS. Moreover, it abolished circulating levels of interleukin (IL)-1β and IL-18 in lipopolysaccharide (LPS)-challenged wild-type mice but not in Nlrp3L351P knock-in mice and ex vivo-stimulated mutant macrophages. These results identify wild-type NLRP3 as the molecular target of MCC950/CRID3 and show that CAPS-related NLRP3 mutants escape efficient MCC950/CRID3 inhibition. Collectively, this work suggests that MCC950/CRID3-based therapies may effectively treat inflammation driven by wild-type NLRP3 but not CAPS-associated mutants.
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http://dx.doi.org/10.1371/journal.pbio.3000354DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6762198PMC
September 2019

IRF2 transcriptionally induces expression for pyroptosis.

Sci Signal 2019 05 21;12(582). Epub 2019 May 21.

Department of Physiological Chemistry, Genentech Inc., South San Francisco, CA 94080, USA.

Gasdermin-D (GSDMD) is cleaved by caspase-1, caspase-4, and caspase-11 in response to canonical and noncanonical inflammasome activation. Upon cleavage, GSDMD oligomerizes and forms plasma membrane pores, resulting in interleukin-1β (IL-1β) secretion, pyroptotic cell death, and inflammatory pathologies, including periodic fever syndromes and septic shock-a plague on modern medicine. Here, we showed that IRF2, a member of the interferon regulatory factor (IRF) family of transcription factors, was essential for the transcriptional activation of A forward genetic screen with -ethyl--nitrosourea (ENU)-mutagenized mice linked IRF2 to inflammasome signaling. expression was substantially attenuated in deficient macrophages, endothelial cells, and multiple tissues, which corresponded with reduced IL-1β secretion and inhibited pyroptosis. Mechanistically, IRF2 bound to a previously uncharacterized but unique site within the promoter to directly drive transcription for the execution of pyroptosis. Disruption of this single IRF2-binding site abolished signaling by both the canonical and noncanonical inflammasomes. Together, our data illuminate a key transcriptional mechanism for expression of the gene encoding GSDMD, a critical mediator of inflammatory pathologies.
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http://dx.doi.org/10.1126/scisignal.aax4917DOI Listing
May 2019

A Map of Toll-like Receptor Expression in the Intestinal Epithelium Reveals Distinct Spatial, Cell Type-Specific, and Temporal Patterns.

Immunity 2018 09 28;49(3):560-575.e6. Epub 2018 Aug 28.

Division of Immunology & Pathogenesis, Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720, USA. Electronic address:

Signaling by Toll-like receptors (TLRs) on intestinal epithelial cells (IECs) is critical for intestinal homeostasis. To visualize epithelial expression of individual TLRs in vivo, we generated five strains of reporter mice. These mice revealed that TLR expression varied dramatically along the length of the intestine. Indeed, small intestine (SI) IECs expressed low levels of multiple TLRs that were highly expressed by colonic IECs. TLR5 expression was restricted to Paneth cells in the SI epithelium. Intestinal organoid experiments revealed that TLR signaling in Paneth cells or colonic IECs induced a core set of host defense genes, but this set did not include antimicrobial peptides, which instead were induced indirectly by inflammatory cytokines. This comprehensive blueprint of TLR expression and function in IECs reveals unexpected diversity in the responsiveness of IECs to microbial stimuli, and together with the associated reporter strains, provides a resource for further study of innate immunity.
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http://dx.doi.org/10.1016/j.immuni.2018.07.016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6152941PMC
September 2018

Caspase-11 auto-proteolysis is crucial for noncanonical inflammasome activation.

J Exp Med 2018 09 22;215(9):2279-2288. Epub 2018 Aug 22.

Department of Physiological Chemistry, Genentech Inc., South San Francisco, CA

Intracellular LPS sensing by caspase-4/5/11 triggers proteolytic activation of pore-forming gasdermin D (GSDMD), leading to pyroptotic cell death in Gram-negative bacteria-infected cells. Involvement of caspase-4/5/11 and GSDMD in inflammatory responses, such as lethal sepsis, makes them highly desirable drug targets. Using knock-in (KI) mouse strains, we herein provide genetic evidence to show that caspase-11 auto-cleavage at the inter-subunit linker is essential for optimal catalytic activity and subsequent proteolytic cleavage of GSDMD. Macrophages from caspase-11-processing dead KI mice ( ) exhibit defective caspase-11 auto-processing and phenocopy and caspase-11 enzymatically dead KI ( ) macrophages in attenuating responses to cytoplasmic LPS or Gram-negative bacteria infection. KI macrophages also fail to cleave GSDMD and are hypo-responsive to inflammasome stimuli, confirming that the GSDMD Asp residue is a nonredundant and indispensable site for proteolytic activation of GSDMD. Our data highlight the role of caspase-11 self-cleavage as a critical regulatory step for GSDMD processing and response against Gram-negative bacteria.
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http://dx.doi.org/10.1084/jem.20180589DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6122968PMC
September 2018

ASC- and caspase-8-dependent apoptotic pathway diverges from the NLRC4 inflammasome in macrophages.

Sci Rep 2018 02 28;8(1):3788. Epub 2018 Feb 28.

Department of Physiological Chemistry, Genentech Inc., South San Francisco, California, USA.

The NLRC4 inflammasome recognizes bacterial flagellin and components of the type III secretion apparatus. NLRC4 stimulation leads to caspase-1 activation followed by a rapid lytic cell death known as pyroptosis. NLRC4 is linked to pathogen-free auto-inflammatory diseases, suggesting a role for NLRC4 in sterile inflammation. Here, we show that NLRC4 activates an alternative cell death program morphologically similar to apoptosis in caspase-1-deficient BMDMs. By performing an unbiased genome-wide CRISPR/Cas9 screen with subsequent validation studies in gene-targeted mice, we highlight a critical role for caspase-8 and ASC adaptor in an alternative apoptotic pathway downstream of NLRC4. Furthermore, caspase-1 catalytically dead knock-in (Casp1 C284A KI) BMDMs genetically segregate pyroptosis and apoptosis, and confirm that caspase-1 does not functionally compete with ASC for NLRC4 interactions. We show that NLRC4/caspase-8-mediated apoptotic cells eventually undergo plasma cell membrane damage in vitro, suggesting that this pathway can lead to secondary necrosis. Unexpectedly, we found that DFNA5/GSDME, a member of the pore-forming gasdermin family, is dispensable for the secondary necrosis that follows NLRC4-mediated apoptosis in macrophages. Together, our data confirm the existence of an alternative caspase-8 activation pathway diverging from the NLRC4 inflammasome in primary macrophages.
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http://dx.doi.org/10.1038/s41598-018-21998-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5830643PMC
February 2018

Tissue-Resident Macrophages Are Locally Programmed for Silent Clearance of Apoptotic Cells.

Immunity 2017 11 14;47(5):913-927.e6. Epub 2017 Nov 14.

Division of Immunology and Pathogenesis, Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720, USA. Electronic address:

Although apoptotic cells (ACs) contain nucleic acids that can be recognized by Toll-like receptors (TLRs), engulfment of ACs does not initiate inflammation in healthy organisms. Here we identified macrophage populations that continually engulf ACs in distinct tissues and found that these macrophages share characteristics compatible with immunologically silent clearance of ACs; such characteristics include high expression of AC recognition receptors, low expression of TLR9, and reduced TLR responsiveness to nucleic acids. Removal of the macrophages from tissues resulted in loss of many of these characteristics and the ability to generate inflammatory responses to AC-derived nucleic acids, suggesting that cues from the tissue microenvironment program macrophages for silent AC clearance. The transcription factors KLF2 and KLF4 control the expression of many genes within this AC clearance program. The coordinated expression of AC receptors with genes that limit responses to nucleic acids might ensure maintenance of homeostasis and thus represent a central feature of tissue macrophages.
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http://dx.doi.org/10.1016/j.immuni.2017.10.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5728676PMC
November 2017

Caspase-11 cleaves gasdermin D for non-canonical inflammasome signalling.

Nature 2015 Oct 16;526(7575):666-71. Epub 2015 Sep 16.

Department of Physiological Chemistry, Genentech Inc., South San Francisco, California 94080, USA.

Intracellular lipopolysaccharide from Gram-negative bacteria including Escherichia coli, Salmonella typhimurium, Shigella flexneri, and Burkholderia thailandensis activates mouse caspase-11, causing pyroptotic cell death, interleukin-1β processing, and lethal septic shock. How caspase-11 executes these downstream signalling events is largely unknown. Here we show that gasdermin D is essential for caspase-11-dependent pyroptosis and interleukin-1β maturation. A forward genetic screen with ethyl-N-nitrosourea-mutagenized mice links Gsdmd to the intracellular lipopolysaccharide response. Macrophages from Gsdmd(-/-) mice generated by gene targeting also exhibit defective pyroptosis and interleukin-1β secretion induced by cytoplasmic lipopolysaccharide or Gram-negative bacteria. In addition, Gsdmd(-/-) mice are protected from a lethal dose of lipopolysaccharide. Mechanistically, caspase-11 cleaves gasdermin D, and the resulting amino-terminal fragment promotes both pyroptosis and NLRP3-dependent activation of caspase-1 in a cell-intrinsic manner. Our data identify gasdermin D as a critical target of caspase-11 and a key mediator of the host response against Gram-negative bacteria.
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http://dx.doi.org/10.1038/nature15541DOI Listing
October 2015

Toll-like receptor-deficient mice reveal how innate immune signaling influences Salmonella virulence strategies.

Cell Host Microbe 2014 Feb;15(2):203-13

Department of Molecular and Cell Biology, Division of Immunology and Pathogenesis, University of California, Berkeley, Berkeley, CA 94720, USA. Electronic address:

Pathogens utilize features of the host response as cues to regulate virulence gene expression. Salmonella enterica serovar Typhimurium (ST) sense Toll-like receptor (TLR)-dependent signals to induce Salmonella Pathogenicity Island 2 (SPI2), a locus required for intracellular replication. To examine pathogenicity in the absence of such cues, we evaluated ST virulence in mice lacking all TLR function (Tlr2(-/-)xTlr4(-/-)xUnc93b1(3d/3d)). When delivered systemically to TLR-deficient mice, ST do not require SPI2 and maintain virulence by replicating extracellularly. In contrast, SPI2 mutant ST are highly attenuated after oral infection of the same mice, revealing a role for SPI2 in the earliest stages of infection, even when intracellular replication is not required. This early requirement for SPI2 is abolished in MyD88(-/-)xTRIF(-/-) mice lacking both TLR- and other MyD88-dependent signaling pathways, a potential consequence of compromised intestinal permeability. These results demonstrate how pathogens use plasticity in virulence strategies to respond to different host immune environments.
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http://dx.doi.org/10.1016/j.chom.2014.01.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3979597PMC
February 2014

Trafficking of endosomal Toll-like receptors.

Trends Cell Biol 2014 Jun 15;24(6):360-9. Epub 2014 Jan 15.

Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA. Electronic address:

Over the past decade we have learned much about nucleic acid recognition by the innate immune system and in particular by Toll-like receptors (TLRs). These receptors localize to endosomal compartments where they are poised to recognize microbial nucleic acids. Multiple regulatory mechanisms function to limit responses to self DNA or RNA, and breakdowns in these mechanisms can contribute to autoimmune or inflammatory disorders. In this review we discuss our current understanding of the cell biology of TLRs involved in nucleic acid recognition and how localization and trafficking of these receptors regulates their function.
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http://dx.doi.org/10.1016/j.tcb.2013.12.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4037363PMC
June 2014

No antigen-presentation defect in Unc93b1(3d/3d) (3d) mice.

Nat Immunol 2013 Nov;14(11):1101-2

Division of Immunology & Pathogenesis, Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, California, USA.

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http://dx.doi.org/10.1038/ni.2733DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3911774PMC
November 2013

UNC93B1 mediates differential trafficking of endosomal TLRs.

Elife 2013 Feb 19;2:e00291. Epub 2013 Feb 19.

Division of Immunology and Pathogenesis, Department of Molecular and Cell Biology , University of California, Berkeley , Berkeley , United States.

UNC93B1, a multipass transmembrane protein required for TLR3, TLR7, TLR9, TLR11, TLR12, and TLR13 function, controls trafficking of TLRs from the endoplasmic reticulum (ER) to endolysosomes. The mechanisms by which UNC93B1 mediates these regulatory effects remain unclear. Here, we demonstrate that UNC93B1 enters the secretory pathway and directly controls the packaging of TLRs into COPII vesicles that bud from the ER. Unlike other COPII loading factors, UNC93B1 remains associated with the TLRs through post-Golgi sorting steps. Unexpectedly, these steps are different among endosomal TLRs. TLR9 requires UNC93B1-mediated recruitment of adaptor protein complex 2 (AP-2) for delivery to endolysosomes while TLR7, TLR11, TLR12, and TLR13 utilize alternative trafficking pathways. Thus, our study describes a mechanism for differential sorting of endosomal TLRs by UNC93B1, which may explain the distinct roles played by these receptors in certain autoimmune diseases.DOI:http://dx.doi.org/10.7554/eLife.00291.001.
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http://dx.doi.org/10.7554/eLife.00291DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3576711PMC
February 2013

The ectodomain of Toll-like receptor 9 is cleaved to generate a functional receptor.

Nature 2008 Dec 28;456(7222):658-62. Epub 2008 Sep 28.

Division of Immunology & Pathogenesis, Department of Molecular and Cell Biology, University of California, Berkeley, 405 Life Sciences Addition, Berkeley, California 94720-3200, USA.

Mammalian Toll-like receptors (TLRs) 3, 7, 8 and 9 initiate immune responses to infection by recognizing microbial nucleic acids; however, these responses come at the cost of potential autoimmunity owing to inappropriate recognition of self nucleic acids. The localization of TLR9 and TLR7 to intracellular compartments seems to have a role in facilitating responses to viral nucleic acids while maintaining tolerance to self nucleic acids, yet the cell biology regulating the transport and localization of these receptors remains poorly understood. Here we define the route by which TLR9 and TLR7 exit the endoplasmic reticulum and travel to endolysosomes in mouse macrophages and dendritic cells. The ectodomains of TLR9 and TLR7 are cleaved in the endolysosome, such that no full-length protein is detectable in the compartment where ligand is recognized. Notably, although both the full-length and cleaved forms of TLR9 are capable of binding ligand, only the processed form recruits MyD88 on activation, indicating that this truncated receptor, rather than the full-length form, is functional. Furthermore, conditions that prevent receptor proteolysis, including forced TLR9 surface localization, render the receptor non-functional. We propose that ectodomain cleavage represents a strategy to restrict receptor activation to endolysosomal compartments and prevent TLRs from responding to self nucleic acids.
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http://dx.doi.org/10.1038/nature07405DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2596276PMC
December 2008

The ubiquitin-editing enzyme A20 restricts nucleotide-binding oligomerization domain containing 2-triggered signals.

Immunity 2008 Mar;28(3):381-90

UCSF Colitis Center, Gastroenterology Division, Department of Medicine, Program in Biomedical Sciences, University of California at San Francisco, San Francisco, CA 94143-0538, USA.

Muramyl dipeptide (MDP), a product of bacterial cell-wall peptidoglycan, activates innate immune cells by stimulating nucleotide-binding oligomerization domain containing 2 (NOD2) -dependent activation of the transcription factor NFkappaB and transcription of proinflammatory genes. A20 is a ubiquitin-modifying enzyme that restricts tumor necrosis factor (TNF) receptor and Toll-like receptor (TLR) -induced signals. We now show that MDP induces ubiquitylation of receptor- interacting protein 2 (RIP2) in primary macrophages. A20-deficient cells exhibit dramatically amplified responses to MDP, including increased RIP2 ubiquitylation, prolonged NFkappaB signaling, and increased production of proinflammatory cytokines. In addition, in vivo responses to MDP are exaggerated in A20-deficient mice and in chimeric mice bearing A20-deficient hematopoietic cells. These exaggerated responses occur independently of the TLR adaptors MyD88 and TRIF as well as TNF signals. These findings indicate that A20 directly restricts NOD2 induced signals in vitro and in vivo, and provide new insights into how these signals are physiologically restricted.
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http://dx.doi.org/10.1016/j.immuni.2008.02.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3606373PMC
March 2008