Cation Channels As Regulators and Effectors of Nlrp3 Inflammasome Signaling and Il-1Β Secretion

CATION CHANNELS AS REGULATORS AND EFFECTORS OF NLRP3 INFLAMMASOME SIGNALING AND IL-1β SECRETION

MICHAEL ALEXANDER KATSNELSON

Submitted in partial fulfillment of the requirements for the degree of

Doctor of Philosophy

Dissertation Advisor: Dr. George R. Dubyak

Department of Pathology

Immunology Training Program

CASE WESTERN RESERVE UNIVERSITY

January, 2016

Case Western Reserve University

School of Graduate Studies

We hereby approve the thesis/dissertation of

Michael Alexander Katsnelson

Candidate for the degree of Doctor of Philosophy

Alan Levine (Committee Chair)

George Dubyak

Corey Smith

Jonathan Smith

Brian Cobb

Amy Hise

Date of Defense

07-30-15

We also certify that written approval has been obtained for any proprietary material contained therein.

Table of Contents

Table of Contents iii

List of Figures v

List of Abbreviations viii

Acknowledgements xiii

Abstract 1

Chapter I: Introduction 3

Part I: IL-1β Signaling in Infection and Autoinflammatory Disease 3

Part II: The Inflammasomes: Macromolecular Complexes That Process IL-1β 9

Part III: NLRP3: A Pattern Recognition Receptor Activated in Response to Changes in Cytosolic Cation Homeostasis 15

Part IV: Regulation of NLRP3 Inflammasome Activation 22

Part V: Consequences of NLRP3 Inflammasome Activation 28

Chapter II: Materials and Methods 36

Chapter III: K+ Efflux Agonists Induce NLRP3 Inflammasome Activation Independently of Ca2+ Signaling 46

Abstract 47

Introduction 48

Results 50

Discussion 87

Chapter IV: Activation or Inhibition of NLRP3 Inflammasome Signaling by Lysosome Destabilization is Coordinated by the Extent

iii of Lysosomal Membrane Permeabilization and Plasma Membrane Cation Channel Activity 95

Abstract 96

Introduction 97

Results 100

Discussion 126

Chapter V: Discussion and Future Directions 136

Bibliography 156

List of Figures

Figure 1.1: Models of inflammasome structure…………………………………..14

Figure 1.2: Proposed upstream activators of NLRP3 and downstream effects of inflammasome activation…………………………………………….….17

Figure 1.3: Positive and negative regulators of proteins within the inflammasome signaling pathway…………………………………………...…….23

Figure 1.4: Stimuli that activate the NLRP3 inflammasome via lysosome permeabilization………………………………………………………………….....32

Figure 1.5: Model of NLRP3 inflammasome activation by lysosome disruption………………………………………………………………………..…..35

Figure 3.1: Nigericin-induced increases in cytosolic [Ca2+] in LPS- primed BMDC occur downstream of the NLRP3 inflammasome/caspase-

1/pyroptotic signaling cascade………………………………………………...…...52

Figure 3.2: NLRP3 inflammasome signaling responses to K+ efflux agonists are dissociated from influx of extracellular Ca2+…………………….…58

Figure 3.3: NLRP3 inflammasome signaling responses to lysosomal

destabilization are dissociated from influx of extracellular Ca2+………………..61

Figure 3.4: NLRP3 inflammasome signaling responses to K+ efflux agonists are dissociated from release of thapsigargin-sensitive intracellular

Ca2+ stores…………………………………………………………………………...65

Figure 3.5: NLRP3 inflammasome signaling responses to K+ efflux agonists are dissociated from changes in cytosolic [Ca2+] in BMDC

primed with TLR2 or TNF receptor agonists………………………………..…..68

Figure 3.6: NLRP3 inflammasome signaling responses to K+ efflux agonists are dissociated from changes in cytosolic [Ca2+] in murine

macrophages………………………………………………………………………..72

Figure 3.7: Increased cytosolic [Ca2+] induced by Ca2+ ionophore or

Ca2+-mobilizing GPCR is not a sufficient signal for NLRP3 inflammasome

activation………...... 76

Figure 3.8: Effects of ionomycin on K+ efflux and cell death in wildtype,

Casp1/11-/-, or Nlrp3-/- BMDC…………………………………………………...78

Figure 3.9: Suppression of nigericin-stimulated NLRP3 inflammasome

signaling by BAPTA can be dissociated from perturbation of Ca2+

signaling……………………………………………………………………………..81

Figure 3.10: Effects of BAPTA on nigericin-induced increases in cytosolic

[Ca2+] in BMDC incubated in Ca2+-containing media………………………….83

Figure 3.11: Suppression of nigericin-stimulated NLRP3 inflammasome

signaling by 2-APB can be dissociated from perturbation of Ca2+

signaling………………...... 85

Figure 4.1: Leu-Leu-O-methyl ester (LLME) induces concentration-

dependent and synchronized increases in lysosomal membrane

permeabilization (LMP) in dendritic cells……………………………………….102

Figure 4.2: NLRP3 inflammasome signaling is activated by low-level

LMP but inhibited by high-level LMP………………………………………...... 106

Figure 4.3: Plasma membrane cation channels and pyroptotic pores are activated by low-level LMP but attenuated by high-level LMP………………..111

Figure 4.4: Influx of extracellular Ca2+ during lysosome destabilization

attenuates LLME-induced NLRP3 inflammasome signaling…………………..116

Figure 4.5: Influx of extracellular Ca2+ during lysosome destabilization

potentiates LLME-induced cell death…………………………………………....120

Figure 4.6: TRPM2 cation channels are not major regulators of LMP-

induced NLRP3 inflammasome signaling in BMDC but contribute to

LMP-activated K+ efflux…………………………………………………………..124

Figure 4.7: Model of biphasic concentration dependence of NLRP3

activation in response to lysosome destabilization……………………………….128

Figure 5.1: Treatment of BMDC with 2-APB causes opening of an ion

channel with the pharmacologic profile of a TRPV family member…………...141

vii

List of Abbreviations

2-APB: 2-aminoethoxydiphenyl borate

AIM-2: Absent in Melanoma 2

ASC: Apoptosis-associated Speck-like Protein Containing a CARD

ATP: Adenosine Triphosphate

BAPTA: 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid

BMDC: Bone Marrow Derived Dendritic Cell

BMDM: Bone Marrow Derived Macrophage

BRCC3: BRCA Containing Complex 3

BRET: Bioluminescence Resonance Energy Transfer

CAPS: Cryopyrin-Associated Periodic Syndromes

CARD: Caspase Recruitment and Interaction Domain

CaSR: Calcium Sensing Receptor

COP: CARD Only Protein

CPPD: Calcium Pyrophosphate Dihydrate

DAMP: Danger Associated Molecular Pattern

DC: Dendritic Cell

viii

DPBA: Diphenylborinic Anhydride

DPTHF: 2,2-diphenyltetrahydrofuran

DsDNA: Double Stranded DNA

DSS: Disuccinimidyl Suberate

ELISA: Enzyme Linked Immunosorbent Assay

ER: Endoplasmic Reticulum

FCAS: Familial Cold Autoinflammatory Syndrome

FITC: Fluorescein Isothiocyanate

GPCR: G Protein Coupled Receptor

GPSM3: G Protein Signaling Modulator 3

HA: Hyaluronan

IAPP: Islet Amyloid Polypeptide

ICAM-1: Intercellular Adhesion Molecule-1

IFI16: Interferon Inducible Protein 16

IFN: Interferon

IKKα: IκB Kinase α

IL-1: Interleukin-1

IL-1R: Interleukin-1 Receptor

IL-1Ra: IL-1 Receptor Antagonist

IL-1RAcP: IL-1 Receptor Accessory Protein

IRAK: Interleukin-1 Receptor Associated Kinase

LDH: Lactate Dehydrogenase

LLME: Leu-Leu-OMe

LMP: Lysosome Membrane Permeabilization

LPS: Lipopolysacharide

LRR: Leucine Rich Repeat Domains

LUBAC: Linear Ubiquitin Assembly Complex

MAP Kinase: Mitogen-Associated Protein Kinase

MSU: Monosodium Urate

MtROS: Mitochondrial Reactive Oxygen Species

MVB: Multivesicular Body

MyD88: Myeloid Differentiation Primary Response Gene 88

NAIP: Neuronal Apoptosis Inhibitory Protein

NF-κB: Nuclear Factor-κB

NLR: NOD Like Receptor

NLRP3: NOD Like Receptor Containing a Pyrin Domain 3

NO/iNOS: Nitric Oxide/Inducible Nitric Oxide Synthase

NOMID: Neonatal Onset Multisystem Inflammatory Disease

PAMP: Pathogen Associate Molecular Pattern

PARP: Poly-ADP Ribose Polymerase

PML: Promyelocytic Leukemia Protein

POP: Pyrin Only Protein

PP2A: Protein Phosphatase 2A

PRR: Pattern Recognition Receptor

PYD: Pyrin Domain

RuRed: Ruthenium Red

SCV: Salmonella Containing Vacuole

T3SS: Type 3 Secretion System

TG: Thapsigargin

TIR: Toll/Interleukin-1 Receptor Homology Domain

TLR: Toll-Like Receptor

TNF: Tumor Necrosis Factor

TRAF: TNF Receptor Associated Factor

TRP: Transient Receptor Potential

TRPM2: Transient Receptor Potential-Melastatin Subfamily 2

TRPV: Transient Receptor Potential-Vanillin Subfamily

TXNIP: Thioredoxin Interacting Protein

UTP: Uridine Triphosphate

VCAM-1: Vascular Cell Adhesion Molecule-1

xii

Acknowledgements:

I would like to thank my mentor, Dr. Dubyak, for giving me the chance to pursue my dream in the MSTP at Case. Thank you for all of the time you have invested in me over the past 4 years. I would also like to thank Dr. Levine and the members of my thesis committee for their time and the guidance that they have given me over the past four years. I would also like to thank my lab mates (Andrea, Hana, Christina, Mausita and

Caroline) for their advice at our Tuesday morning lab meetings and for making the lab a fun environment in which to work. I could not have come this far without the support of my family. Thank you for nurturing my interest in science and constantly pushing me to excel.

xiii

Cation Channels as Regulators and Effectors of NLRP3 Inflammasome Signaling and IL-1β Secretion

Abstract

MICHAEL ALEXANDER KATSNELSON

IL-1β is a pro-inflammatory cytokine that plays a crucial role in the systemic

response to infection. Dysregulated IL-1β signaling is involved in multiple genetically

inherited, autoimmune, and chronic inflammatory diseases. IL-1β is expressed as a pro-

cytokine which must be processed by cellular proteases into the mature form to mediate

signaling. Several Pattern Recognition Receptors (PRRs) are capable of inducing

processing of pro-IL-1β by the protease caspase-1 via stimulating the formation of

cytosolic macromolecular complexes called inflammasomes.

NLRP3 is a unique inflammasome-forming PRR because it senses a wide variety

of cellular stressors including bacterial pore forming toxins (which alter cytosolic cation

homeostasis) and crystalline particulates (which disrupt lysosome integrity). Although the ligand of NLRP3 remains unknown, we and others have suggested that efflux of cytosolic

K+ is the common upstream event mediating NLRP3 inflammasome assembly. Several

recent studies have utilized pharmacologic approaches to implicate Ca2+ signaling in

NLRP3 activation. In the first part of this dissertation, I hypothesized that increases in

cytosolic [Ca2+] act synergistically with efflux of cytosolic K+ to stimulate NLRP3

inflammasome complex assembly. However, we observed that increases in cytosolic

[Ca2+] are not required for activation of NLRP3 inflammasome signaling in response to strong K+ efflux agonists (the K+ ionophore nigericin and opening of the P2X7 cation channel by extracellular ATP) and inhibit NLRP3 inflammasome signaling in response to

lysosome permeabilization.

I also sought to elucidate the mechanism by which lysosome destabilization

stimulates NLRP3 inflammasome assembly. In the second part of this dissertation, I

hypothesized that lysosome destabilizers activate NLRP3 inflammasome signaling by

causing the opening of channels/pores in the plasma membrane which then mediate

efflux of cytosolic K+. I found that collapse of lysosome integrity causes a change in

permeability of the plasma membrane to K+ and Ca2+ upstream of inflammasome

activation. This disruption of cation homeostasis then drives NLRP3 inflammasome

signaling as well as an inflammasome-independent necrotic cell death which is dependent

upon influx of extracellular Ca2+. My studies have forged a link between two different

forms of cell stress that are both sensed by NLRP3: changes in cytosolic cation

homeostasis and lysosome permeabilization.

Chapter 1: Introduction

Part I IL-1β Signaling in Infection and Autoinflammatory Disease

Functions of IL-1β in the Response to Infection

The IL-1 cytokines are one of the principal mediators of the systemic

inflammatory response to infection. IL-1 acts on the central nervous system to induce fever and also activates the hypothalamic-pituitary-adrenal axis. Stimulation of the adrenal cortex by Adrenocorticotropic Hormone (ACTH) causes the release of cortisol, a hormone which has an overall suppressive effect on the immune response(1). IL-1 stimulates the production of acute phase proteins in the liver; these include molecules which amplify the humoral arm of the immune response (including C-reactive protein, mannose binding lectin, and complement components) as well as α1-antitrypsin, which limits tissue damage by extracellular proteases released from inflammatory cells(1). IL-1 signaling increases synthesis of cyclooxygenase-type 2 (COX2), phospholipase A (type

2), and inducible nitric oxide synthase (iNOS)(2). Increased expression of these molecules stimulates the release of prostaglandin-E2 (PGE-2), platelet activating factor and nitric oxide (NO). Release of these inflammatory mediators causes reduced pain threshold, vasodilation and hypotension(2). IL-1 also acts as a pro-angiogenic factor and stimulates the formation of new blood vessels(2). IL-1 signaling upregulates expression of adhesion molecules (including ICAM-1 on mesenchymal cells and VCAM-1 on endothelial cells) and chemokines, which amplify the recruitment of leukocytes to sites of infection(1). IL-1 increases the lifespan and stimulates effector functions of neutrophils and macrophages, while promoting differentiation of bone marrow stem cells into myeloid progenitor cells(1). IL-1 signaling is essential for differentiation of the TH17

subset of helper T cells; mice with a deficiency of the IL-1R1 fail to produce IL-17 upon challenge with antigens(3).

The IL-1 Signaling System

The IL-1 family of cytokines includes two closely related members: IL-1α and IL-

1β(3). These two inflammatory signaling molecules likely arose as a result of a gene duplication event approximately 300 million years ago and their functions have diverged over the course of evolutionary history(4). IL-1α is expressed constitutively in a wide variety of cell types (including myeloid cells, epithelium, and keratinocytes) and is released from cells during necrotic cell death(1). The full length p33 form of IL-1α is capable of mediating signaling. However, IL-1α may be cleaved by calpain proteases into a mature p17 form, which has a 40-fold higher binding affinity for the IL-1R compared to the full length (p33) form of the cytokine(5). IL-1β expression is induced in myeloid leukocytes in response to stimulation of TLRs, TNFR1, or IL-1R1. These pro- inflammatory receptors stimulate expression of pro-IL-1β protein by activating NF-κB signaling(1). Pro-IL-1β must be cleaved into a mature p17 form by inflammatory or apoptotic caspases (caspase-1 or caspase-8) or neutrophil enzymes (proteinase-3 and elastase) in order to mediate signaling(2,6).

The IL-1R type 1 is a transmembrane receptor consisting of an extracellular region made up of three immunoglobulin-like domains and an intracellular TIR domain, which is essential for interaction with the adaptor protein MyD88. Binding of IL-1α or

IL-1β to the extracellular portion of the receptor causes the interaction of IL-1R1 with IL-

1R accessory protein (IL-1RAcP) and this complex then mediates intracellular signaling

events, via recruitment of cytosolic MyD88(1). Interaction of MyD88 with cytosolic TIR domains leads to the formation of a multiprotein complex (which includes MyD88,

IRAK1, IRAK2, IRAK4 and TRAF6) that mediates activation of NF-κB and MAPK signaling cascades, resulting in transcription of a variety of pro-inflammatory gene products(2).

IL-1R2 is a decoy receptor which negatively regulates inflammatory signaling; it contains a truncated cytosolic region which lacks the TIR domain and is thus incapable of mediating signaling. IL-1R2 acts as a molecular sink by binding to IL-1α and IL-1β with high affinity. It also exerts a dominant negative effect on intracellular signaling by forming a complex with IL-1RAcP(1). IL-1R2 may be localized to the plasma membrane or may be secreted into the extracellular medium(1). IL-1 receptor antagonist (IL-1Ra) is a secreted protein that acts as an antagonist of IL-1 signaling. IL-1Ra binds to IL-1R1 with a higher affinity than IL-1α or IL-1β. However, interaction of IL-1Ra and IL-1R1 fails to cause recruitment of the IL-1RAcP and thus does not result in activation of downstream inflammatory signaling(1).

Dysregulated Overproduction of IL-1β Underlies Pathology of Cryopyrin-Associated

Periodic Fever Syndromes

Cryopyrin-associated periodic syndromes (CAPS) are three autoinflammatory disorders which are driven by mutation of the NLRP3 gene on chromosome 1. These disorders are inherited in an autosomal dominant manner with variable penetrance(7).

Patients who inherit mutant alleles of NLRP3 (which encodes a protein regulating IL-1β processing and release, as described below) inappropriately produce high amounts of IL-

1β and IL-18(7). Excessive IL-1β production is thought to drive most of the pathology

observed in CAPS patients(8). The three syndromes represent a spectrum of severity.

Familial cold autoinflammatory syndrome (FCAS) is the mildest in severity. Upon

exposure to cold temperatures, patients spontaneously develop fever, urticarial rash, and

arthralgias. Attacks usually resolve within 24 hours(8). Muckle-Wells syndrome (MWS) is a more severe phenotype than FCAS; patients present with the autoinflammatory symptoms mentioned above plus sensorineural hearing loss and secondary amyloidosis with nephropathy. Febrile episodes occur at irregular intervals and precipitating factors may not always be identified(8). Neonatal-onset multisystem inflammatory disease

(NOMID) is the most severe of the CAPS. Aside from urticarial rash and fever, patients often exhibit impaired growth, facial abnormalities (frontal bossing, protruding eyes, saddle-shaped nose), chronic meningitis, hearing loss, and cerebral atrophy. NOMID often causes premature death(8).

Excessive IL-1β Production Contributes to the Pathology of Autoimmune Disease and

Sterile Inflammation

More moderate overproduction of IL-1β (compared to that observed in CAPS) plays a critical role in the pathogenesis of several autoimmune diseases. Rheumatoid arthritis is a chronic autoimmune disease which primarily affects synovial joints.

Analysis of synovial fluid from affected joints typically reveals an inflammatory infiltrate which is composed predominantly of polymorphonuclear (PMN) cells(9). Gradual infiltration of PMN cells into the joint space results in erosion of neighboring bone and articular cartilage, narrowing of the joint space, and eventually causes severely limited

range of motion and permanent deformation of the joint(10). Along with TNFα and IL-6,

IL-1 signaling plays a crucial role in driving articular damage in rheumatoid arthritis. IL-

1 is expressed predominantly by macrophages in the inflamed joint and levels of IL-1 in

the synovial fluid correlate with severity of articular disease(11). IL-1α/β promote the

production of collagenase and stromelysins by synovial fibroblasts; these enzymes promote the breakdown of articular cartilage. IL-1 cytokines also drive activation of osteoclasts to promote bone resorption and decrease proteoglycan synthesis by articular cartilage. Taken together, these effects of IL-1 serve to cause degradation of articular cartilage and breakdown of bone near the joint space(12). IL-1 also stimulates production of chemokines by articular fibroblasts and causes upregulated expression of endothelial- cell adhesion molecules, promoting the further infiltration of leukocytes into the joint space(12).

Metabolic syndrome X is characterized by elevated fasting glucose levels, elevated triglyceride levels, abdominal obesity, and hypertension. It is prevalent in 22% of the US adult population and substantially increases the risk of developing type 2 diabetes and coronary vessel disease(13). Metabolic syndrome X is characterized by a state of sterile chronic inflammation and IL-1β contributes to the pathogenesis of this condition(13,14). Recent reports indicate that the saturated fatty acids palmitate and stearate, which are found in elevated amounts in patients with metabolic syndrome, are capable of stimulating IL-1β release from macrophages by disrupting integrity of lysosomes and inducing production of ROS(15). Saturated fatty acids were also shown to be capable of upregulating expression of pro-IL-1β by activating TRL2/TLR4 signaling(16). Other reports indicate that IL-1β is released by pancreatic macrophages

under conditions of chronic hyperglycemia. Elevated glucose levels cause release of islet

amyloid polypeptide (IAPP) from pancreatic β cells. IAPP has a propensity to misfold

and form aggregates composed of long fibrils when released into the extracellular space.

Internalization of IAPP fibrils by pancreatic macrophages results in IL-1β release, which amplifies pancreatic inflammation and contributes to death of insulin-producing β

cells(14,17). A recent study utilizing insulin-resistant obsese mice expressing human

IAPP found progressive amyloid accumulation within the pancreas and increased levels

of IL-1β within the islets of Langerhans. Administration of IL-1Ra resulted in improved

glucose tolerance and enhanced insulin release from pancreatic islets of transgenic mice

expressing hIAPP(18).

Upon release, IL-1β mediates substantial changes in systemic metabolism. In a

model of obesity induced insulin resistance, mice with deficiency of IL-1β or IL-1R1

were protected from hyperglycemia. IL-1β contributes to insulin resistance of adipocytes

by downregulating expression of Insulin Receptor Substrate (IRS)-1, which resulted in

reduced activation of Protein Kinase B (PKB) in response to insulin and decreased

translocation of the Glut4 glucose transport channel to the plasma membrane(19).

Treatment with IL-1β also enhanced insulin resistance in murine hepatocytes by

inhibiting phosphorylation of Akt in response to insulin treatment(20). IL-1β stimulated de novo lipogenesis in hepatocytes and caused accumulation of triglycerides within murine liver cells(21). This phenotype of triglyceride accumulation within the liver

(steatosis) is present in many individuals with metabolic syndrome and has been termed

Non-Alcoholic Fatty Liver Disease (NAFLD), a condition associated with chronic

8 inflammation of the liver (steatohepatitis) and progressive cirrhosis (gradual replacement of hepatocytes with fibrous tissue)(22).

Aside from its role in metabolic disease, IL-1β has been implicated in a variety of inflammatory conditions driven by sterile crystalline particulates. Gout is an autoinflammatory disease characterized by elevated levels of uric acid, a byproduct of purine metabolism. When present at high concentrations, uric acid may form crystals of monosodium urate (MSU). MSU crystals are deposited in joints and drive an autoinflammatory arthritis(23). Pseudogout is another crystal-induced arthropathy driven by deposition of calcium pyrophosphate dihydrate (CPPD) crystals in the synovial space(24). Martinon et al. demonstrated that both MSU and CPPD crystals are phagocytized by macrophages and stimulate the release of mature IL-1β from these cells(25). Mice with a deficiency of IL-1R1 demonstrated reduced neutrophil infiltration in response to peritoneal injection of MSU or CPPD crystals. Martinon proposed that IL-

1β released from synovial monocytes/macrophages drives joint inflammation by activating a pro-inflammatory gene expression program in synovial fibroblasts, as described above(25).

Part II The Inflammasomes: Macromolecular Complexes That Process IL-1β

Pattern Recognition Receptors of Innate Immunity

The innate immune system relies on five main classes of receptors to recognize

Pathogen Associated Molecular Patterns (PAMPs). Toll-like Receptors (TLRs) are transmembrane receptors that are capable of recognizing a variety of PAMPs via

extracellular Leucine Rich Repeat (LRR) domains(26). Ligands of the TLRs include LPS from Gram-negative bacteria (TLR4), peptidoglycan from Gram-positive bacteria

(TLR2), double stranded RNA from viruses (TLR3), single stranded viral RNA (TLR7 and 8), and unmethylated CpG DNA found in prokaryotes and DNA viruses (TLR9)(27).

Once bound to ligand, TLRs mediate signaling through cytosolic TIR domains via the

adaptor molecules MyD88 or TRIF(26). C-type lectin receptors are transmembrane molecules containing at least one C-type lectin-like domain (CTLD). This receptor family includes members such as Dectin-1, Dectin-2 and Mincle. The CTLDs typically recognize pathogen-derived carbohydrate moieties such as β-glucans or α-mannans(28).

Activating C-type lectin receptors signal through cytosolic ITAM-like motifs (or recruit

ITAM-containing adaptor molecules such as the Fc receptor γ chain) to activate spleen tyrosine kinase (Syk) signaling. This signaling pathway leads to downstream activation of

NF-κB or the MAPK cascade and triggers cellular responses such as phagocytosis, DC maturation, respiratory burst or cytokine production. C-type lectin receptors may also inhibit inflammatory functions; receptors containing cytosolic ITIM domains recruit tyrosine phosphatases (SHP-1 and SHP-2) to modulate the signaling of other PRRs(28).

Two groups of PRRs lack transmembrane domains, are predominantly localized in the cytosol and recognize PAMPs present in the intracellular space. Nucleotide- binding oligomerization domain (NOD)-like receptors contain an amino-terminal protein- protein interaction domain, a central nucleotide binding domain, and a C-terminal LRR domain(29). The NLRs may be divided into two broad subfamilies: NLRC and NLRP.

NLRCs contain one or two amino terminal CARD domains, which interact with caspases or kinases to mediate downstream signaling. NLRP family members contain an amino

terminal Pyrin domain (PYD), which recruits the adaptor protein Apoptosis-associated

Speck-like Protein Containing a CARD (ASC) to mediate inflammatory signaling(29).

RIG-I-Like Receptors (RLRs) consist of RIG-I, MDA5, and LGP2. This group of PRRs detects viral RNA present in the cytosol. The RLRs possess a central helicase domain and a C-terminal domain which are responsible for ligand recognition(30). RIG-I and MDA5

have two N-terminal CARD domains which mediate downstream signaling. Upon

binding of viral RNA, RIG-I and MDA5 recruit the downstream adaptor protein MAVS

via CARD-CARD interactions. RLR-MAVS complexes then activate IKK signaling via

TRAF3 or TRAF6 to initiate transcription of IFNs and pro-inflammatory cytokines(30).

Lastly, PRRs of the PYHIN family contain a DNA-binding HIN domain and a PYRIN

domain. This family includes AIM2 (which recognizes pathogen-derived dsDNA in the

cytosol) and IFI16 (which recognizes pathogen-derived dsDNA in the nucleus)(31).

Activation of Certain PRRs Causes Formation of Macromolecular IL-1β Processing

Platforms

Activation of several of the NLRs or the HIN-200 domain-containing protein

AIM2 results in the formation of cytosolic macromolecular complexes called

inflammasomes. These complexes consist of a PRR (NLRP1, NLRP3, NLRC4 or AIM2),

the adaptor protein ASC, and the protease caspase-1(32). Activation of PRR by its cognate ligand stimulates association of the PRR with the adaptor protein ASC via PYD-

PYD interactions and causes aggregation of cellular ASC into a large speck-like

structure. ASC then recruits pro-caspase-1 to the speck via CARD-CARD interactions(32). When pro-caspase-1 proteins are brought into close proximity within the

ASC speck, they cleave each other to form mature caspase-1 enzyme. Caspase-1 then mediates the proteolytic processing and release of IL-1β. The 33kDa pro-IL-1β protein is cleaved to generate the mature 17kDa form the cytokine, which is released from the cell via the process of non-canonical secretion(32).

The murine genome contains three paralogs of NLRP1 (a,b, and c). NLRP1b causes inflammasome assembly after being proteolytically cleaved by anthrax lethal toxin. NLRP1b contains a CARD domain and mediates IL-1β processing independently of ASC. However, presence of ASC potentiates IL-1β release in response to anthrax lethal toxin(33). NLRP3 mediates inflammasome formation in response to a wide variety of pathogen-derived components including bacterial hemolysins and pore-forming toxins.

NLRP3 is also activated by a variety of sterile Danger-Associated Molecular Patterns

(DAMPs) including extracellular ATP and crystalline particulates (including silica, asbestos, monosodium urate, and cholesterol)(34). The NLRC4 inflammasome is activated in response to bacterial flagellin or proteins of the Type III Secretion System

(T3SS)/Type IV Secretion System (T4SS). Members of the NAIP protein family serve as upstream sensors of these bacterial components; NAIP5 and NAIP6 recognize cytosolic flagellin, NAIP1 recognizes the needle protein of the T3SS and NAIP2 recognized the inner rod protein of the T3SS(35). NLRC4 contains a CARD domain and interacts directly with pro-caspase-1 to mediate autocatalytic processing of this enzyme. However,

IL-1β release in response to NLRC4 activators is enhanced in the presence of ASC.

NLRC4 activation reduces cytoskeletal rearrangement to prevent further phagocytosis of bacteria, enhances ROS production to eliminate intracellular bacteria, and reduces cell motility to prevent dissemination of pathogens(33). AIM2 is a protein composed of a

HIN-200 (capable of detecting pathogen-derived dsDNA in the cytosol) and a PYD

capable of interacting with ASC. AIM2 is capable of mediating IL-1β production in

response to numerous intracellular bacteria (including Francisella tularensis, Listeria

monocytogenes, and Streptococcus pneumoniae) and dsDNA viruses (including

cytomegalovirus and vaccinia)(33).

Models of Inflammasome Complex Structure

Considerable debate exists about the nature of the inflammasome complex

structure (Figure 1.1). The spoked-wheel model holds that inflammasomes have a

structure similar to that of the apoptosome, a wheel-like particle composed of cytochrome

c/Apaf-1 complexes that mediates processing of pro-caspase-9 during apoptosis. It has

been proposed that PRRs form a seven-spoked wheel with the sensory LRR domains at

the rim and pro-caspase-1 localized to the hub. In a series of elaborate Bioluminescence

Resonance Energy Transfer (BRET) experiments, Compan et al. demonstrated that

NLRP3 self-associates at baseline, with N- and C-termini of neighboring PRRs in close proximity. This group proposed that activation of NLRP3 in response to hypotonic stress induces bending of the LRR domain away from the central hub, causing separation of N- and C- termini and cessation of BRET signal(36). Several recent reports have proposed a

“branching tree” model of inflammasome activation. Cai et al. found that the ASC PYD possesses prion-like properties; aggregated ASC-PYD mediated the conversion of inactive soluble ASC into a high molecular weight filament capable of mediating caspase-1 processing. Mutations in the ASC PYD that interfere with filament formation also inhibit the ability of ASC to mediate downstream signaling. Finally, the authors

Figure 1.1: Models of inflammasome structure. A) Layered speck model. B) Branching tree filament model. C) Apoptosome-like model.

elegantly demonstrated that an ASC construct in which the PYD has been replaced with a fungal prion domain retains the ability to process caspase-1(37). Lu et al. proposed a

“tree-like” model of inflammasome activation: PRRs form a heptameric root that nucleates the polymerization of a prion-like ASC filament (the trunk) and pro-caspase-1

“branches” extend laterally from the ASC trunk(38). A third model of inflammasome structure has been termed the “layered speck.” This model is based on high-resolution immunofluorescence microscopy of macrophages infected with S. typhimurium, which is known to stimulate activation of the NLRC4 inflammasome. Man et al. observed that the

ASC speck is composed of an outer ring of ASC, an inner ring of PRRs (NLRC4 and

NLRP3), and pro-caspase-1 localized to the center of the structure(39).

Part III NLRP3: A Pattern Recognition Receptor Activated in Response to Changes in

Cytosolic Cation Homeostasis

Proposed Mechanisms of NLRP3 Activation

NLRP3 is a unique PRR because it may be activated in response to a wide variety of structurally diverse stimuli. These include bacterial hemolysins/pore forming toxins, extracellular ATP, a variety of crystalline particulates, and amyloid plaques(34). Several theories have been proposed to explain how this wide range of stimuli causes activation of a single PRR (Figure 1.2). Activation of NLRP3 in response to phagocytized particulates requires disruption of lysosome membrane integrity and release of lysosomal cathepsin proteases into the cytosol(40,41). Lysosome membrane disruption by hypotonic stress or the lysosomotropic agent leu-leu-oMe (LLME) also results in activation of

NLRP3(40). Bafilomycin A (an inhibitor of lysosome acidification) and pharmacologic

inhibitors of cathepsin activity attenuate NLRP3 activation in response to

particulates(40). In a recent study, Okada et al. proposed that lysosome disruption causes

the release of lysosomal Ca2+ stores and activation of CaMKII. This results in activation of Tak1-JNK signaling (a MAPK pathway), which is required for NLRP3 activation in response to lysosome rupture(42). However, these findings are complicated by the fact

that CaMKII activity is necessary for NF-κB mediated cytokine production in response to

TLR stimulation(43). Therefore, inhibition of CaMKII activity may interfere with the ability of macrophages to produce pro-IL-1β.

Nearly all NLRP3 activators stimulate production of mitochondrial reactive oxygen species (mtROS) and scavenging of mitochondrial superoxide free radicals attenuates NLRP3 inflammasome activation(44). Several mechanisms have been proposed for how ROS activate NLRP3. ROS may modify an endogenous molecule to generate a DAMP which may be recognized by NLRP3. One such molecule is

Thioredoxin-Interacting Protein (TXNIP), which interacts with reduced thioredoxin

(TXN) in unstimulated cells. ROS generated in response to NLRP3 agonists cause oxidation of TXN and dissociation of TXNIP from TXN. Free TXNIP then associates with NLRP3 and causes the receptor to adopt an active conformation(45). TXNIP is required for NLRP3 activation in response to ER stress(46). However, Masters et al. found that TXNIP is dispensable for NLRP3 activation in response to ATP, amyloid and monosodium urate crystals(17). A second possibility is that mtROS may directly oxidize

NLRP3. NLRP3 is predicted to contain a disulfide bond within the PYD which may be modified by ROS. Oxidized NLRP3 is predicted to adopt an active conformation(47).

Figure 1.2: Proposed upstream activators of NLRP3 and downstream effects of inflammasome activation.

However, a recent study has demonstrated that the effect of mtROS on NLRP3 may be

indicrect. Heid et al. observed that generation of mtROS leads to lysosomal membrane

permeabilization (LMP)(48). LMP and the associated release of lysosomal hydrolases

into the cytosol may then enhance NLRP3 inflammasome complex assembly.

Other studies have suggested that NLRP3 is activated in response to recognition

of mitochondrial components by the PRR. One of these components is proposed to be

oxidized mtDNA. Depletion of macrophage mtDNA by treatment with ethidium bromide

inhibits inflammasome activation and NLRP3 agonists (nigericin and extracellular ATP) have been shown to cause release of oxidized mtDNA into the cytosol(49,50). Upon its release, oxidized mt DNA interacts specifically with NLRP3 and introduction of exogenous oxidized mtDNA into the cytosol of macrophages stimulates inflammasome activation(49). However, DNA-deficient mitochondria have abnormal expression of cytochrome c and abnormal respiratory function, which complicate the interpretation of the experiments utilizing ethidium bromide to deplete mtDNA(50). Also, NLRP3-/-

macrophages do not demonstrate diminished IL-1β release in response to oxidized

mtDNA(49).

Another proposed NLRP3 ligand is cardiolipin, a phospholipid found in the inner

mitochondrial membrane. Iyer et al. found that cardiolipin interacts with the LRR domain

of NLRP3 and inflammasome assembly could be recapitulated in lysates of LPS-primed

macrophages treated with cardiolipin-containing liposomes(51). Inhibition of cardiolipin synthesis by treatment with palmitate (which functions as an inhibitor of enzymes involved in the cardiolipin synthesis pathway) and siRNA knockdown of cardiolipin

synthase inhibited IL-1β release in response to silica and extracellular ATP by

monocytes/macrophages(51).

Unified Mechanism of NLRP3 Inflammasome Activation: Role of Cytosolic Potassium

Efflux

Our group and others have proposed efflux of cytosolic K+ as the common trigger

for NLRP3 activation(52). All known activators of NLRP3 (including lysosome- destabilizing crystalline particulates) cause a reduction in cytosolic [K+] and inhibition of

cytosolic K+ efflux (by increasing extracellular [K+]) abrogates NLRP3 inflammasome

assembly/IL-1β release(53,54). When inflammasome components are exposed to a low

K+ medium in vitro, the proteins self-assemble to form a functional inflammasome

complex(54). Munoz-Planillo et al. demonstrated that exposure of macrophages to low

+ + + [K ] extracellular medium ([K ]ex≤0.5mM) caused a slow efflux of cytosolic K from the cells, which was sufficient to stimulate IL-1β release. Treatment of macrophages with ouabain, an inhibitor of the Na+/K+ ATPase, also caused a reduction in cytosolic [K+] and

IL-1β release(53). Munoz-Planillo et al. also argue that perturbation of mitochondria is

not required for NLRP3 activation. Treatment of macrophages with extracellular ATP

(which causes opening of the nonselective cation channel P2X7) and gramicidin (a

bacterial pore-forming toxin) stimulated inflammasome activation and IL-1β release.

Both stimuli also caused in increase in Oxygen Consumption Rate (OCR) and

Extracellular Acidification Rate (ECAR), which indicate mitochondrial stress. The

gramicidin and ATP-induced increases in OCR and ECAR were eliminated by

preincubation with ouabain, while inflammasome activation/IL-1β release in response to

these agonists were not diminished in the presence of ouabain. From these observations,

the authors argue that mitochondrial dysfunction observed after treatment with

gramicidin and extracellular ATP occurs as a result of increased Na+/K+ ATPase function,

as the cell attempts to compensate for perturbations in cation homeostasis caused by

insertion of pores or opening of nonselective cation channels(53). The authors also found

that increased mtROS production was not required for NLRP3 activation. The bacterial

pore-forming toxin gramicidin activated NLRP3 signaling without causing an increase in

mtROS. Furthermore, the authors did not observe inflammasome activation when

macrophages were stimulated with rotenone (which increases mtROS by acting as an

inhibitor of the electron transport chain) and hydrogen peroxide(53).

Proposed Role of Increased Cytosolic [Ca2+] in NLRP3 Inflammasome Activation.

Several recent reports have proposed a role for increases in cytosolic [Ca2+] in

NLRP3 inflammasome activation. Many NLRP3 activators (including nigericin,

extracellular ATP, crystalline particulates and the lysosomotropic peptide LLME) cause

increases in cytosolic [Ca2+](55). Brough et al. demonstrated that IL-1β release in

response to nigericin and extracellular ATP was inhibited by BAPTA-AM, a chelator of

cytosolic Ca2+(56). NLRP3 inflammasome activation in response to these agonists was

2+ also suppressed by 2-APB (which inhibits ER Ca release by blocking IP3R and Store-

Operated Calcium Entry Channels) and U73122 (an inhibitor of Phospholipase C, an

2+ enzyme that mediates ER Ca release by generating IP3)(57). Several studies have demonstrated that Ca2+ mobilizing G-Protein Coupled Receptors (GPCRs) also activate

NLRP3 signaling; these include the extracellular Ca2+-sensing receptor (CaSR) and the

related family number GPRC6A(58,59). However, the Ca2+ ionophore ionomycin (which

triggers large increases in cytosolic [Ca2+]) is not capable of causing NLRP3

inflammasome activation(57). Taken together, these findings suggest that increases in

cytosolic [Ca2+] are not sufficient to activate NLRP3 inflammasome signaling. We and

others have proposed that increases in cytosolic [Ca2+] may act in concert with efflux of

cytosolic K+ to mediate NLRP3 inflammasome activation.

TRP Channels May Mediate Cation Flux Upstream of NLRP3 Activation

Transient Receptor Potential Channels (TRPs) are a family of nonselective cation channels which mediate cytosolic K+ efflux and influx of extracellular Ca2+(60). Several

TRPs have recently been implicated in mediating cation flux to cause activation of the

NLRP3 inflammasome. A recent study has proposed that opening of TRPM2 is required

for inflammasome activation in response to crystalline particulates. TRPM2 is activated

upon binding to intracellular ADP-ribose (ADPR), a molecule generated in response to mitochondrial stress (61). Zhong et al. proposed that lysosome destabilization by silica

particles induces mitochondrial damage and concurrent generation of ADPR. Binding of

ADPR to the cytosolic domain of TRPM2 causes opening of the channel, which

stimulates inflammasome assembly by mediating influx of extracellular Ca2+ (62).

Compan et al. have implicated TRPV2 and TRPM7 in NLRP3 inflammasome activation induced by hypotonic stress. Treatment of macrophages with pharmacologic TRP channel inhibitors suppressed NLRP3-dependent caspase-1 processing/IL-1β release in response to hypotonic stress(36). These findings indicate that TRP channels may serve as conduits to mediate cation flux in response to NLRP3 activators that are not expected to

directly alter cytosolic cation levels (such stimuli include crystalline particulates or the

lysosomotropic agent LLME). Lysosome destabilization in response to treatment of

myeloid cells with particulates results in the release of lysosome cathepsin proteases into

the cytosol; these proteases may mediate changes in cytosolic cation levels by i)

proteolytically opening TRPs by cleaving off the gating domain of the channel(s) ii)

causing mitochondrial damage, which results in the generation of ROS and opening of

ROS-sensitive TRPM2 channels in the plasma membrane.

Part IV: Regulation of NLRP3 Inflammasome Activation (Figure 1.3)

COPs and POPs:

COPs are proteins containing a single CARD domain, which function as dominant

negative inhibitors of inflammasome signaling by binding to the CARD domain of pro-

caspase-1 and preventing autocatalytic activation of this enzyme. COPs include the

proteins ICEBERG, Pseudo-ICE, and INCA(63). These proteins are expressed in myeloid

cells and expression is increased in response to LPS (ICEBERG and Pseudo-ICE) or

IFNγ (INCA). Overexpression of COPs in THP-1 monocytes inhibits caspase-1

dependent IL-1β release(64,65). POPs are proteins containing a single PYRIN domain,

which inhibit inflammasome signaling by interacting with the PYDs of NLRPs/AIM2

and ASC. The human genome encodes four POPs: POP1, POP2, POP3 and POP4(63).

POP2 is expressed in myeloid cells and expression is upregulated in response to pro-

inflammatory stimuli such as LPS and TNF(63). Overexpression of POP2 inhibits

NLRP3 inflammasome activation by interfering with the formation of ASC

Figure 1.3: Positive and negative regulators of proteins within the inflammasome signaling pathway.

23 aggregates(66). COPs and POPs are encoded within primate genomes, but are absent in rodents(63).

Regulation of NLRP3

NLRP3 requires a “priming” signal, which may be delivered via a variety of receptors which mediate signaling via MyD88 or TRIF. These receptors include the

TLRs, TNFR1, IL-1R1, and NOD2. These receptors mediate NF-κB signaling, which upregulates expression of NLRP3 and other inflammasome components over a timescale of several hours(1). Recently, Fernandes-Alnemri et al. characterized a rapid priming pathway (occurring within 10 min of TLR stimulation) which is independent of new protein synthesis. This group observed that simultaneous activation of MyD88 signaling and NLRP3 causes rapid inflammasome assembly in a process dependent on IRAK4 and

IRAK1 kinase activity. The authors also demonstrated that IRAK1 associates with

NLRP3 upon TLR stimulation(67).

Nitric oxide (NO) is produced by macrophages in response to IFNγ release by T cells; NO is able to mediate post-translational modification of proteins by oxidatively modifying the thiol group of cysteine side chains(68). A recent study proposed that

NLRP3 activity is regulated by s-nitrosylation; Hernandez-Cuellar et al. found that activation of iNOS in macrophages causes s-nitrosylation of NLRP3 and caspase-1(69).

Treatment of macrophages with the NO donor SNAP inhibited NLRP3 inflammasome activation in response to nigericin and ATP(69). NLRP3 is also regulated via ubiquitination. Py et al. found that the LRR domains of NLRP3 are basally ubiquitinated

and that inhibiting deubiquitinase activity with the small molecule G5 abrogated inflammasome activation in response to nigericin/silica particles(70). The authors demonstrated that the deubiquitinase BRCC3 bound to and mediated deubiquitination of

NLRP3 upon LPS priming; genomic knockdown of BRCC3 inhibited NLRP3 inflammasome activation(70).

In addition to post-translational modification, NLRP3 activity is also regulated by a variety of binding partners. A complex of G Protein Signaling Mediator 3

(GPSM3)/HSPA8 interacts with the LRR domains of NLRP3 and maintains the inactive conformation of the receptor. Mice with a genomic deficiency of GPSM3 demonstrated enhanced IL-1β release in response to NLRP3 activators (extracellular ATP and alum particles). The authors proposed that LPS priming licenses NLRP3 for activation by reducing expression of GPSM3 and thus liberating the LRR domains for interaction with ligand(71).

Regulation of ASC

ASC is constitutively expressed by macrophages/DCs. In resting myeloid cells

ASC localizes to the nucleus, where it is found in a complex with Promyelocytic

Leukemia protein (PML) and IKKα(72,73). Association with PML maintains the nuclear localization of ASC(72). The ASC/IKKα complex translocates to the perinuclear area following stimulation of myeloid cells with “signal 1” TLR ligands in a process that requires IKKi; the kinase activity of IKKα maintains ASC in an inactive state outside the

nucleus. Upon stimulation with a “signal 2” NLRP3 activator such as extracellular ATP,

ASC is dephosphorylated by Protein Phosphatase 2A (PP2A) and dissociates from

IKKα(73).

Whereas IKKα kinase activity inhibits formation of inflammasome complexes,

the kinase activities of Syk and JNK have been demonstrated to promote inflammasome

activation in macrophages. ASC is phosphorylated at Tyr144 in a Syk/JNK-dependent

manner and phosphorylation of this residue is required for ASC speck formation in

response to activators of the NLRP3 and AIM2 inflammasomes(74). ASC activity is also

regulated by ubiquitination. Upon activation of NLRP3, ASC is linearly ubiquitinated by

the Linear Ubiquitin Assembly Complex (LUBAC), an enzyme complex which forms

linear (M1) ubiquitin chains on target substrates(75). Linear ubiquitination of ASC is required for NLRP3-mediated inflammasome activation(76).

Regulation of Pro-IL-1β

There is no detectable pro-IL-1β expression in myeloid cells under basal conditions. Expression of pro-IL-1β is upregulated in response to TLR stimulation and downstream NF-κB signaling(1). Recently, it has been found that pro-IL-1β is also subject to post-translational modification. Upon LPS priming, pro-IL-1β is found in a complex with pro-caspase-1, pro-caspase-8, RIPK1, RIPK3 and the deubiquitinase

A20(77). The deubiquitinase activity of A20 negatively modulates pro-IL-1β processing/release; A20-deficient macrophages demonstrated spontaneous processing/release of IL-1β in response to TLR4 priming, but in the absence of a “signal

2” inflammasome-activating stimulus. In A20-deficient macrophages, pro-IL-1β is

modified by attachment of K63-linked and unanchored poly-ubiquitin chains in a RIPK3-

mediated process. Ubiquitination of pro-IL-1β in A20-deficient cells promoted the

processing/release of the mature form of the cytokine even in the absence of an NLRP3-

activating “signal 2” stimulus(77). The authors also observe that NLRP3 activation

stimulates recruitment of more A20 to the active inflammasome complex; here, the

deubiquitinase activity of this enzyme may serve to downregulate the inflammatory

response by preventing IL-1β processing/release(77).

Negative Regulation of Inflammasomes by Autophagy

Autophagy (“self-eating”) is a stress response in which dysfunctional organelles

or misfolded/polyubiquitinated proteins are targeted for degradation within intracellular double membrane-bound structures called autophagosomes. Autophagosome formation is initiated by a regulatory complex containing the proteins beclin-1 and Vps-34. Once formed, the autophagosome fuses with lysosomes and the enclosed proteins/organelles are degraded by lysosomal enzymes(78). Ubiquitin-binding proteins such as p62 and

NBR1 act as autophagy adaptors and selectively target polyubiquitinated ligands to the autophagosome(79). Experimental evidence indicates that autophagy acts as a negative regulator of inflammasome signaling. Depletion of the autophagy regulatory proteins beclin-1 and LC3B (which are critical for autophagosome formation) in murine macrophages enhances caspase-1 processing and release of IL-1β/IL-18 in response to

NLRP3 agonists(78).

Stimulation of various TLRs (“signal 1”) has been demonstrated to upregulate

autophagy(80). Additionally, treatment of myeloid cells with inflammasome activators

(‘signal 2”) also promotes the induction of autophagy. Activation of the NLRP3 and

AIM2 inflammasomes causes nucleotide exchange on the small GTPase RalB; nucleotide

exchange on RalB drives autophagosome formation(81). Upon activation of the

inflammasome, ASC is modified by conjugation with K63-polyubiquitin chains.

Ubiquitinated ASC is recognized by the autophagy adaptor p62, which targets ASC

aggregates to autophagosomes(81). Thus, induction of autophagy by TLR signaling and

inflammasome activation serves as a negative feedback mechanism to degrade activated

inflammasome complexes and prevent release of excessive amounts of IL-1β/IL-18. It has also been proposed that autophagy removes damaged mitochondria from the cell and prevents the release of NLRP3 activators such as mtROS or mitochondrial components

(mtDNA, cardiolipin)(78).

Part V: Consequences of NLRP3 Inflammasome Activation (Figure 1.2)

Non-Classical Export of Cytokines and DAMPs

Activation of the NLRP3 inflammasome results in caspase-1 dependent export of

a variety of intracellular molecules into the extracellular space. These exported molecules

include IL-1β, IL-18, IL-1α, fibroblast growth factors, and HMGB1 (a nuclear protein

which acts as a DAMP when released into the extracellular compartment)(82). IL-1β

lacks a signal sequence and thus cannot be exported via the classical pathway of

ER→Golgi→secretory vesicles. Several mechanisms have been proposed for how

28 caspase-1 mediates the non-classical secretion of inflammasome-dependent cytokines and

DAMPs(82). IL-1β/IL-18 may be exported by secretory lysosomes, which mediate the rapid release of pro-inflammatory mediators from activated myeloid cells. Several studies have proposed that active caspase-1 and pro-IL-1β are transported from the cytosol into secretory lysosomes, where caspase-1 mediates cytokine processing. The secretory lysosomes then fuse with the plasma membrane to release processed IL-1β(83,84).

Others have proposed that IL-1β/IL-18 release is mediated by shedding of plasma- membrane derived microvesicles into the extracellular environment. Studies performed with several types of myeloid cells and microglia indicate that caspase-1 and IL-1β accumulate at cytosolic microdomains of the plasma membrane; these phosphatidylserine-enriched microdomains then form blebs that are eventually scissioned away from the cell(82,85). Secretion of inflammasome-dependent cytokines can also be accomplished by the release of exosomes, which are derived from intracellular multivesicular bodies (MVBs). MVBs are formed from endosomes in which the limit membrane has undergone invagination to form multiple intra-luminal vesicles that contain cytosolic proteins. MVBs may fuse with the plasma membrane and deliver intraluminal vesicles to the extracellular compartment; once released into the extracellular space, the intra-luminal vesicles are called exosomes(82). Qu et al. reported that secretion of caspase-1 and IL-1β from murine macrophages strongly correlated with the NLRP3- dependent release of MHC-II containing exosomes. The authors concluded that inflammasome complexes and IL-1β may be localized to MVBs and released via exosomes(86).

Pyroptosis: An Inflammatory Cell Death Pathway

Pyroptosis is a programmed cell death pathway initiated by the activity of pro-

inflammatory caspases (caspases-1, 4, 11). Pyroptotic cell death is induced by activated

inflammasomes and is mediated by the proteolytic activity of caspase-1(87). Although

caspase enzymatic activity is required for this cell death pathway, proteolytic processing

of pro-caspase-1 into mature caspase-1 is not necessary(88). Caspase-1 activity causes

the opening of large non-selective pores in the plasma membrane; these pores are

permeable to cations (Na+, K+, Ca2+) and small molecular weight dyes (such as propidium iodide), but remain impermeable to higher molecular weight species such as PEG-

1450(89). Extracellular ions and water enter cells via pyroptotic pores, causing cell

swelling and eventual lysis. The end result of pyroptosis is the release of intracellular contents into the extracellular space, where many of these cellular components act as

DAMPs to signal to neighboring immune cells(90). Pyroptotic cells undergo DNA damage and stain positive by TUNEL assay. However, extensive DNA laddering and breakdown of the nuclear membrane (characteristic of apoptotic cell death) does not occur during pyroptosis(87). PARP is an enzyme which is activated during un- programmed necrosis; active PARP consumes NAD+ and decreases cellular ATP stores.

PARP is inactivated by caspase activity during apoptotic cell death, when ATP is

required for execution of the programmed cell death pathway. PARP is active in cells

undergoing pyroptosis; however, PARP activity is not required for pyroptosis as PARP-/-

macrophages are still able to undergo pyroptosis in response to NLRC4 inflammasome

activation by S. typhimurium(87).

Recent studies indicate that some effector functions of inflammasome activation

may be mediated entirely by pyroptotic cell death rather than release of IL-1β/IL-18.

Clearance of flagellin-expressing S. typhimurium infection in mice was found to be entirely dependent on pyroptosis and independent of inflammatory cytokines(91). S. typhimurium infects macrophages and DCs; within these cells the bacteria reside within a protected vacuole structure. Activation of pyroptosis in macrophages/DCs causes the elimination of this protected intracellular niche. S. typhimurium bacteria are expelled into the extracellular space, where they are phagocytosed by neutrophils and destroyed by

ROS generated via the NADPH oxidase system(91). Another study has demonstrated that depletion of helper T cells during chronic HIV infection occurs principally as a result of pyroptosis. The DNA sensor IFI16 is activated by binding to reverse-transcribed HIV ssDNA. IFI16 mediates activation of caspase-1, death of CD4+ T cells by pyroptosis, and release of pro-inflammatory cytokines that promote chronic inflammation observed in

HIV-infected patients(92,93).

Lysosome Destabilizers Activate a Caspase-Independent Necrotic Cell Death Pathway

As described above, the NLRP3 inflammasome may be activated by a variety of lysosome destabilizing compounds including crystalline particulates, saturated fatty acids and lysosomotropic small molecules such as LLME (Figure 1.4). Aside from activating inflammasome signaling, lysosome destabilizers also induce a caspase-independent form of cell death. This cell death pathway has been termed “lysosome membrane permeabilization (LMP)-induced cell death.” LMP-induced cell death is initiated by

Figure 1.4: Stimuli that activate the NLRP3 inflammasome via lysosome permeabilization. Lysosome membrane permeabilization occurs in response to accumulation of free fatty acids (such as palmitate and oleate) within lysosomes.

Particulates (alum, silica, asbestos, monosodium urate, cholesterol crystals) are thought to tear through the lysosome membrane with jagged crystal edges. Small lysosomotropic molecules such as LLME are polymerized into detergent-like polypeptides by lysosomal hydrolases and these detergent molecules then disrupt integrity of the lysosome membrane.

permeabilization of the lysosome membrane and release of lysosomal enzymes into the

cytosol. Specifically, lysosomal cathepsin proteases have been implicated as mediators of

this cell death pathway. Inhibition of cathepsin activity by pharmacologic agents or by

overexpression of cystatins (cellular protease inhibitors localized in the cytosol)

abrogates LMP-induced cell death(94). The mechanisms/signaling pathways by which

cathepsin protease activity causes cell death remain incompletely characterized.

Lysosome membrane permeabilization has recently been demonstrated to initiate

epithelial cell death during the process of mammary gland involution. Upon cessation of

lactation, STAT3 activity causes mammary epithelial cells to phagocytize Milk Fat

Globules (MFGs) remaining in the mammary gland lumen. The internalized MFGs are transported to lysosomes, where acid lipases degrade milk triglycerides into free fatty acids. One of the free fatty acids released as a result of MFG degradation is oleic acid, which permeabilizes the lysosomal membrane and causes release of lysosomal cathepsins into the cytosol(95). Cytosolic cathepsins cause death of mammary epithelial cells by mediating proteolytic degradation of as-yet unidentified targets.

Hypotheses of Dissertation Research

In the first part of this dissertation, I hypothesized that increases in cytosolic

[Ca2+] act synergistically with efflux of cytosolic K+ to stimulate NLRP3 inflammasome

complex assembly. Increased cytosolic [Ca2+] may activate NLRP3 inflammasome

signaling by i) opening Ca2+-activated K+ channels to mediate K+ efflux, ii) promoting

assembly of ASC aggregates or iii) enhancing release of IL-1β via nonclassical secretion.

I observed that increases in cytosolic [Ca2+] are not necessary for NLRP3 activation in

response to opening of ATP-gated P2X7 channels, the K+ ionophore nigericin, or the

lysosome destabilizing agent leu-leu-oMe (LLME). Ca2+ mobilizing G-Protein Coupled

Receptors (GPCRs) failed to induce rapid activation of the NLRP3 inflammasome. The intracellular Ca2+ chelator BAPTA and the channel inhibitor 2-APB, widely used in previous studies to test the role of Ca2+ signaling in NLRP3 activation, inhibit

inflammasome assembly independently of their effects on cytosolic [Ca2+]. In the second

part of this dissertation, I sought to elucidate the mechanism by which lysosome

destabilization stimulates NLRP3 inflammasome assembly. I hypothesized that lysosome

destabilizers activate NLRP3 inflammasome signaling by causing the opening of

channels/pores in the plasma membrane which then mediate efflux of cytosolic K+

(Figure 1.5). I found that collapse of lysosome integrity causes a change in permeability

of the plasma membrane to K+ and Ca2+ upstream of NLRP3 inflammasome activation,

leading to altered cytosolic cation levels. This disruption of cation homeostasis then

drives NLRP3 inflammasome signaling (via efflux of cytosolic K+) as well as inflammasome-independent necrotic cell death (which is dependent upon influx of extracellular Ca2+). These studies have forged a link between two different forms of cell

stress that are both sensed by NLRP3: changes in cytosolic cation homeostasis and

lysosome permeabilization.

Figure 1.5: Model of NLRP3 inflammasome activation by lysosome disruption.

Permeabilization of the lysosome membrane causes release of lysosomal cathepsins into

the cytosol. Cathepsins proteolytically modify as-yet unidentified cation channels

(TRPs?) in the plasma membrane. Channel opening mediates K+ efflux to activate the

NLRP3 inflammasome.

Chapter 2: Materials and Methods

Reagents

Key reagents and their sources were as follows: Escherichia coli LPS serotype

O1101:B4 (List Biological Laboratories), Pam3CSK4 (InvivoGen), murine rTNF-α

(PeproTech), murine rM-CSF (PeproTech), nigericin (Sigma-Aldrich), ATP (Sigma-

Aldrich), ionomycin (LC Laboratories), N-formyl-Met-Leu-Phe (Sigma-Aldrich), UTP

(Sigma-Aldrich), H-Leu-Leu-OMe·HBr (Bachem), lidocaine (Sigma-Aldrich), R568

(Tocris Bioscience), thapsigargin (LC Laboratories), disuccinimidyl suberate (DSS;

Sigma-Aldrich), BAPTA-AM (Molecular Probes), 2-APB (Tocris Bioscience), anti– caspase-1 (p20) mouse mAb (Casper-1) (Adipogen), anti-NLRP3 rat mAb (Clone

768319; R&D Systems), anti-ASC rabbit polyclonal Ab (N-15), anti-β actin goat polyclonal Ab (C-11), and all HRP-conjugated secondary Abs (Santa Cruz

Biotechnology), murine IL-1β ELISA kit (R&D Systems), fluo-4–acetoxymethyl ester

(fluo-4-AM; Life Technologies), probenecid (Sigma-Aldrich), propidium iodide (Life

Technologies), lactate dehydrogenase (LDH) cytotoxicity detection kit (Roche), FITC- conjugated 10kDa dextran (Life Technologies). Anti–IL-1β mouse mAb was provided by the Biological Resources Branch, National Cancer Institute, Frederick Cancer Research and Development Center (Frederick, MD).

Murine Models

Wild-type (WT) C57BL/6 mice were purchased from Taconic. Mice lacking both caspase-1 and caspase-11 on a C57BL/6 background (Casp1/11−/−) have been previously

described(96). Nlrp3−/− mice were provided by Dr. A. Hise (Case Western Reserve

University). All experiments and procedures involving mice were approved by the

Institutional Animal Care and Use Committee of Case Western Reserve University. Bone

marrow–derived DC (BMDC) and bone marrow–derived macrophages (BMDM) were

isolated from 9- to 12-wk-old mice. Mice were euthanized by CO2 inhalation. Bones from TRPM2-knockout mice were shipped in serum-free DMEM on ice from Penn State

Hershey Children’s Hospital to Case Western Reserve University by overnight courier

service. Femurs and tibiae were removed and briefly sterilized in 10% ethanol. PBS was

used to wash out the marrow plugs. Bone marrow cells were resuspended in DMEM

(Sigma-Aldrich) supplemented with 10% bovine calf serum (HyClone Laboratories), 100

U/ml penicillin, 100 μg/ml streptomycin (Invitrogen), 2 mM l-glutamine (Lonza), and either 4% J558L cell-conditioned medium (which contains the GM-CSF necessary for

BMDC differentiation) or 20 ng/ml rM-CSF (necessary for BMDM differentiation). Bone marrow cells were plated onto 150-mm plastic petri dishes and cultured in the presence of

10% CO2.

For BMDC, the nonadherent cell population was removed on day 3 postisolation,

centrifuged at 300 × g for 5 min at room temperature, resuspended in fresh GM-CSF

supplemented medium, and replated onto a 150-mm petri dish. At 5 d postisolation, the loosely adherent BMDC were collected, pelleted by centrifugation (300 × g, 5 min), and

resuspended to 1 × 106 cells/ml in fresh growth medium and plated in six-well (2

ml/well) tissue culture plates for Western blot assays, 12-well (1 ml/well) plates for K+

atomic absorption assays, and 24-well (0.5 ml/well) plates for IL-1β ELISA, fluo-4

assays of cytosolic [Ca2+] changes, propidium2+ influx assays, LDH release assays of

cytotoxicity, or assays of lysosome integrity using FITC-dextran loaded cells. All assays

of BMDC function were performed between days 7 and 10 postisolation. For BMDM,

33% of the initial volume of growth medium was removed on day 3 postisolation and

replaced with fresh DMEM containing 20 ng/ml rM-CSF. At 7 d postisolation, the adherent BMDM were detached using PBS supplemented with 5 mM EGTA and 4 mg/ml lidocaine, pelleted by centrifugation (300 × g, 5 min), and resuspended to 1 × 106

cells/ml in fresh growth medium described above. The BMDM were then plated into 6-,

12-, or 24-well plates as described for the BMDC.

Priming and Stimulation of BMDC and BMDM

Prior to experimental treatments, BMDC-containing TC plates were centrifuged at

300 × g for 5 min to prevent loss of the loosely adherent cells; this step was not required for the highly adherent BMDM. The growth/differentiation medium was removed from

BMDC- or BMDM-containing plates and replaced with DMEM (10% bovine calf serum,

penicillin, streptomycin, and l-glutamine) supplemented with either 1 μg/ml LPS, 2 μg/ml

Pam3CSK4, or 100 ng/ml TNF-α as “signal 1” priming stimuli to induce NF-κB–

dependent upregulation of pro–IL-1β and NLRP3 expression. The cells were treated with

these priming stimuli for 4 h at 37°C. Plates with primed BMDC were again centrifuged

at 300 × g for 5 min before further manipulation. For either BMDC or BMDM cultures,

the priming medium was aspirated after 4 h and replaced with either Ca2+-containing

balanced salt solution (BSS) (130 mM NaCl, 4 mM KCl, 1.5 mM CaCl2, 1 mM MgCl2,

25 mM Na HEPES, 5 mM d-glucose, 0.1% BSA [pH 7.4]), Ca2+-free BSS (130 mM

NaCl, 4 mM KCl, 300 μM EGTA, 1 mM MgCl2, 25 mM Na HEPES, 5 mM d-glucose,

+ 0.1% BSA [pH 7.4]), or high K BSS (134 mM KCl, 1.5 mM CaCl2, 1 mM MgCl2, 25

mM Na HEPES, 5 mM d-glucose, 0.1% BSA [pH 7.4]). BMDC and BMDM in BSS were

preincubated for 5 min at 37°C and then stimulated with 10 μM nigericin, 5 mM ATP,

various concentrations of LLME, or various concentrations of Ca2+-mobilizing agents for

various times as indicated in specific experiments. Where indicated, signal 1–primed

BMDC and BMDM were treated for 30 min with 300 nM thapsigargin in Ca2+-free BSS

to deplete endoplasmic reticulum (ER) Ca2+ stores and transferred to either fresh Ca2+-

free BSS or Ca2+-containing BSS prior to stimulation with nigericin or ATP.

ELISA Analysis of IL-1β Release

Signal 1–primed BMDC or BMDM in 24-well plates (5 × 105 cells/0.5 ml

BSS/well) were stimulated with nigericin, ATP, LLME, or Ca2+-mobilizing agents at

37°C. After the indicated time interval, extracellular medium was removed from each

well and centrifuged at 10,000 × g for 15 s to pellet detached cells. The cell-free supernatants were then assayed for murine IL-1β by sandwich ELISA (R&D Systems)

according to the manufacturer’s protocol.

Western Blot Analysis of Caspase-1 Processing/Release and IL-1β Processing/Release

Signal 1–primed BMDC in six-well plates (2 × 106 cells/1 ml BSS/well) were

stimulated with nigericin, ATP, LLME, or Ca2+-mobilizing agents at 37°C. After 30 min,

the extracellular medium was removed from each well and briefly centrifuged to pellet

detached cells. The cell-free supernatant was transferred to a new tube, the extracellular

proteins were concentrated by trichloroacetic acid precipitation, and the precipitated

proteins were processed for SDS-PAGE as previously described(97). Any detached cells in the pellet from each extracellular medium sample were dissolved in RIPA buffer and added back to the original well containing the adherent BMDC to generate whole-cell lysate samples as described previously(97). Cell lysates and matching extracellular medium samples were subjected to SDS-PAGE and transferred to a polyvinylidene fluoride membrane for Western blot analysis. Primary Abs were used at the following concentrations: 1 μg/ml for caspase-1, 5 μg/ml for IL-1β, 0.4 μg/ml for ASC, 4µg/ml for

NLRP3, and 0.2µg/ml for β actin. HRP-conjugated secondary Abs were used at a concentration of 0.13 μg/ml. Chemiluminescent images of Western blots were developed and saved using a FluorChem E image processor (Cell Biosciences).

Assay of ASC Oligomerization Using DSS-Crosslinked Detergent-Insoluble Lysate

Fractions

After stimulation, the extracellular medium from each well was removed, centrifuged at 10,000 × g for 15 s to pellet any detached BMDC, and the cell-free supernatant was aspirated. The adherent BMDC in each well were washed with 1 ml ice- cold PBS prior to preparation of whole-cell detergent lysates by addition of 150 μl lysis buffer (0.5% sodium deoxycholate, 0.1% SDS, 1% Igepal CA-630 in PBS [pH 7.4], plus protease inhibitor mixture), scraped with a rubber policeman to fully detach the cells, and

incubated on ice for 5 min. This cell lysate was pooled with the detached cell pellet, and

the resulting whole-cell lysate was incubated for an additional 15 min on ice. The cell lysates were then separated into detergent-soluble and detergent-insoluble fractions by centrifugation at 15,000 × g for 15 min at 4°C. SDS sample buffer was added to detergent-soluble fractions for extraction at 100°C for 5 min. The detergent-insoluble lysate pellet was washed twice with 200 μl ice-cold PBS and then suspended in 200 μl

PBS containing 2 mM DSS (from a stock 20 mM DSS solution in DMSO). The resuspended detergent-insoluble fractions were incubated with DSS for 30 min at room temperature, repelleted by centrifugation at 8000 × g for 15 min at room temperature, and the DSS supernatant solution was removed. The DSS-treated pellets were suspended in

SDS-PAGE buffer and extracted at 100°C for 5 min. The DSS-treated fractions were resolved by SDS-PAGE, transferred to polyvinylidene fluoride membrane, and analyzed by anti-ASC Western blotting.

Atomic Absorption Spectroscopy for Measurement of K+ Efflux

Signal 1–primed BMDC in 12-well plates (106 cells/1 ml BSS/well) were

stimulated for indicated times with nigericin, ATP, LLME, or Ca2+-mobilizing agents at

37°C. After indicated time interval, the extracellular medium was removed and centrifuged at 10,000 × g for 15 s to pellet detached cells. Adherent cells were briefly

+ washed with K -free BSS (135 mM sodium gluconate, 1.5 mM CaCl2, 1 mM MgCl2, 25

mM HEPES [pH 7.4]) to remove any residual K+ from BSS treatment medium. The wash

solution from each well was used to resuspend the detached cell pellet from that well and

this suspension was recentrifuged at 10,000 × g for 15 s to repellet the cells. One

milliliter 10% nitric acid was used to resuspend the detached cell pellet and this was then

added back to the corresponding well containing the adherent BMDC. This 1-ml nitric

acid extract was supplemented with an additional 1 ml 10% nitric acid and the BMDC

were extracted into the resulting 2 ml nitric acid volume for 2 h to ensure adequate

extraction of cellular K+ content. The nitric acid lysates were then transferred into 2-ml

microcentrifuge tubes and the amount of elemental potassium in each lysate was

quantified using a 50 AA atomic absorption spectrometer (Agilent Technologies).

Propidium2+ Influx Assay of Plasma Membrane Permeabilization

Signal 1–primed BMDC or BMDM in 24-well plates (5 × 105 cells/well) were

briefly washed with PBS prior to the addition of 0.5 ml BSS (Ca2+-containing or Ca2+-

free) supplemented with 2 μg/ml propidium iodide to each well. The plate was placed

into a Synergy HT plate reader (BioTek) preheated to 37°C. Baseline fluorescence (540

nm excitation → 620 nm emission at 30-s intervals) was recorded for 5 min. Cells were

then stimulated with 10 μM nigericin, 5 mM ATP, various concentrations of LLME, or 3

μM ionomycin for indicated time and changes in 540ex→620em fluorescence were recorded at 30-s intervals. Assays were terminated by permeabilization of the BMDC with 1% Triton X-100 to quantify maximum fluorescence of the propidium2+/DNA complexes. Where indicated, cells were treated with 300 nM thapsigargin in Ca2+-free

BSS for 30 min to deplete ER Ca2+ stores prior to addition of nigericin or ATP. The

agonist-induced increases in fluorescence at each time point were normalized as a

percentage of the maximum fluorescence measured after complete permeabilization of all

BMDC/BMDM within the well with 1% Triton X-100.

Fluo-4–Based Assay of Cytosolic [Ca2+]

Signal 1–primed BMDC or BMDM in 24-well plates (5 × 105 cells/well) were

briefly washed with PBS prior to the addition of 0.5 ml BSS supplemented with 1 μM

fluo-4-AM and 2.5 mM probenecid per well. After incubation at 37°C for 45 min, each

well was briefly washed with PBS prior to the addition of a fresh 0.5-ml aliquot of BSS

(either Ca2+-containing or Ca2+-free) supplemented with 2.5 mM probenecid. The plate was placed into the Synergy HT reader preheated to 37°C. Baseline fluorescence (485 nm excitation → 528 nm emission at 30-s intervals) was recorded for 10 min. Cells were then stimulated with 10 μM nigericin, 5 mM ATP, various concentrations of LLME, or

2+ indicated concentrations of Ca -mobilizing agents and changes in 485ex→528em

fluorescence were recorded at 30-s intervals. Where indicated, cells were treated with 300

nM thapsigargin in Ca2+-free BSS for 30 min to deplete ER Ca2+ stores prior to addition

of nigericin or ATP. Assays were terminated by permeabilization of the cells with 1%

2+ Triton X-100 in the presence of excess CaCl2 to quantify the maximum Ca -dependent

fluorescence (Fmax) of the fluo-4 indicator dye. The wells were then supplemented with

15 mM EGTA/50 mM Tris to chelate Ca2+ and quantify the minimum Ca2+-independent

fluorescence of fluo-4 (Fmin). The Fmax and Fmin values were used to calculate the

2+ cytosolic [Ca ] corresponding to changes in 485ex→528em fluorescence of fluo-4 within

intact cells as described by Tsien and colleagues(98).

Assay of Cytotoxicity (LDH Release)

Signal 1–primed BMDC in 24-well plates (5 × 105 cells/0.5 ml BSS/well) were

stimulated with 10 μM nigericin or various concentrations of LLME at 37°C for indicated

times. Extracellular medium was removed from each well and centrifuged at 10,000 × g

for 15 s to pellet detached cells. The cell-free supernatants were then assayed for LDH

enzyme activity using an LDH cytotoxicity detection kit (Roche) according to the

manufacturer’s protocol. The released LDH was normalized to total LDH content

measured in 1% Triton X-100–permeabilized samples of BMDC.

Assay of Lysosomal Membrane Permeabilization

A previously described method (99) was adapted for on-line analysis of lysosomal membrane permeabilization during stimulation of BMDC with LLME or nigericin.

BMDC in 24-well plates (5 × 105 cells/well) were incubated in complete DMEM

supplemented with 100 µg/ml FITC-conjugated dextran (10 kDa). After overnight

(~15h) loading, the FITC-dextran-supplemented DMEM was replaced and the cell monolayers washed once with 1 ml PBS. The FITC-dextran-loaded BMDC were then primed with LPS (1µg/ml in complete DMEM; 0.5 ml/well) for 4 h. The priming medium was the replaced with 0.5 ml BSS (Ca2+-containing or Ca2+-free) per well and the plate

transferred to the Synergy HT reader preheated to 37°C. Baseline fluorescence (485em

nm→528ex) was recorded for 5 min at 30-s intervals. Cells were then stimulated with 10

µM nigericin or indicated concentrations of LLME for 30 min with 485ex→528em fluorescence recorded at 30-s intervals. Immediately afterwards, phase-contrast and

epifluorescence images of the BMDC in each well were recorded using a Zeiss Axiovert

25 Microscope equipped with a 485 nm/540 nm filter set, QCam1394 digital camera, and

QCapturePro imaging software (QImaging).

Data Processing and Analysis

All experiments were repeated 2–10 times with separate BMDC or BMDM

preparations. Figures illustrating Western blot results are from representative

experiments. Figures illustrating quantified changes in IL-1β secretion, intracellular K+

content, cytosolic [Ca2+], propidium2+ influx, LDH activity, or lysosome permeabilization

represent the means (±SE) from 2 to 10 independent experiments. Quantified data were

statistically evaluated by one-way ANOVA with a Bonferroni posttest using Prism 3.0

software.

Chapter 3: K+ Efflux Agonists Induce NLRP3 Inflammasome Activation Independently of Ca2+ Signaling.

Portions of this chapter have been published in:

Katsnelson, M. A., Rucker, L. G., Russo, H. M., and Dubyak, G. R. (2015) K+ Efflux Agonists Induce NLRP3 Inflammasome Activation Independently of Ca2+ Signaling. The Journal of Immunology 194, 3937-3952.

Abstract:

Perturbation of intracellular ion homeostasis is a major cellular stress signal for

activation of NLRP3 inflammasome signaling that results in caspase-1–mediated

production of IL-1β and pyroptosis. However, the relative contributions of decreased

cytosolic K+ concentration versus increased cytosolic Ca2+ concentration ([Ca2+]) remain

disputed and incompletely defined. We investigated roles for elevated cytosolic [Ca2+] in

NLRP3 activation and downstream inflammasome signaling responses in primary murine

dendritic cells and macrophages in response to two canonical NLRP3 agonists (ATP and

nigericin) that facilitate primary K+ efflux by mechanistically distinct pathways or the

lysosome-destabilizing agonist Leu-Leu-O-methyl ester. The study provides three major findings relevant to this unresolved area of NLRP3 regulation. First, increased cytosolic

[Ca2+] was neither a necessary nor sufficient signal for the NLRP3 inflammasome cascade during activation by endogenous ATP-gated P2X7 receptor channels, the exogenous bacterial ionophore nigericin, or the lysosomotropic agent Leu-Leu-O-methyl ester. Second, agonists for three Ca2+-mobilizing G protein–coupled receptors (formyl

peptide receptor, P2Y2 purinergic receptor, and calcium-sensing receptor) expressed in

murine dendritic cells were ineffective as activators of rapidly induced NLRP3 signaling

when directly compared with the K+ efflux agonists. Third, the intracellular Ca2+ buffer,

BAPTA, and the channel blocker, 2-aminoethoxydiphenyl borate, widely used reagents

for disruption of Ca2+-dependent signaling pathways, strongly suppressed nigericin-

induced NLRP3 inflammasome signaling via mechanisms dissociated from their

canonical or expected effects on Ca2+ homeostasis. The results indicate that the ability of

K+ efflux agonists to activate NLRP3 inflammasome signaling can be dissociated from

changes in cytosolic [Ca2+] as a necessary or sufficient signal.

Introduction:

Interleukin-1β is a primary proinflammatory cytokine that activates the acute phase response, induces fever, promotes proliferation of neutrophils in the bone marrow, and stimulates adherence of leukocytes to the walls of blood vessels(100). Production of biologically active IL-1β requires its proteolytic processing (maturation) by caspase-1.

Caspase-1 activation per se involves autocatalytic processing regulated by assembly of inflammasome signaling complexes(34). One major type of inflammasome consists of the cytosolic pattern recognition receptor NLRP3, the adaptor protein ASC, and procaspase-1(32,34). Upon activation, NLRP3 monomers oligomerize into a ring-like structure that recruits ASC monomers to induce formation of ASC filaments or specks(37,38). In turn, ASC filaments/specks recruit multiple procaspase-1 monomers to facilitate the induced proximity required for autocatalytic proteolysis into the 10- and 20- kDa subunits that assemble into the highly active tetramers of caspase-1. Active caspase-

1 mediates both the proteolytic maturation of IL-1β and its release via noncanonical secretion(32,34). Caspase-1 also induces pyroptosis, a regulated cell death pathway characterized by the permeabilization of the plasma membrane that facilitates collapse of ionic and osmotic homeostasis and eventual cell lysis(101).

The assembly of NLRP3 inflammasomes can be activated by various exogenous stimuli, including extracellular ATP (via opening of P2X7 nonselective cation channel receptors), small microbial ionophores such as nigericin or gramicidin, and large bacterial pore-forming protein toxins(102,103). All of these NLRP3-activating stimuli directly

perturb the permeability of the plasma membrane to K+ with consequent reduction in

cytosolic K+ concentration ([K+]). Multiple studies have identified decreased cytosolic

[K+] as a necessary signal for the induction of NLRP3 inflammasome assembly by those

stimuli that directly target plasma membrane permeability(52). Importantly, Muñoz-

Planillo et al. (53) reported that the ability of particulate stimuli, such as monosodium urate, silica, and aluminum hydroxide, or small molecule lysosomotropic molecules, such as Leu-Leu-O-methyl ester (LLME), to activate NLRP3 inflammasomes secondary to

lysosomal destabilization also requires efflux of K+. However, it remains unclear whether

decreased cytosolic [K+] is a sufficient ionic signal in addition to being a necessary signal. In this regard, other studies have implicated elevations in cytosolic Ca2+

concentration ([Ca2+]) in the NLRP3 inflammasome activation response to several

stimuli, including nigericin and ATP-gated P2X7 receptor channels. Several reports indicated that caspase-1 activation and IL-1β release are suppressed in macrophages loaded with BAPTA, a chelator of cytosolic Ca2+ (56,104). Murakami et al. (57) found that NLRP3 inflammasome signaling in response to nigericin and ATP was markedly

2+ attenuated by inhibitors of phospholipase C (U73122), IP3-gated Ca release channels

(xestospongin C), or store-operated Ca2+ entry (2-aminoethoxydiphenyl borate [2-APB]).

Finally, agonists for the calcium-sensing G protein–coupled receptor (GPCR; CaSR) and

C5a complement GPCR, which stimulate phospholipase C–mediated increases in

cytosolic [Ca2+], have been reported to induce NLRP3 inflammasome–dependent IL-1β

release from murine or human monocytes/macrophages(58,59,105).

These observations suggest that increased cytosolic [Ca2+] may activate NLRP3

inflammasome signaling either independently of, or synergistically with, decreased

cytosolic [K+]. However, no previous studies have directly measured changes in both cytosolic [Ca2+] and [K+] in myeloid leukocytes under the experimental conditions routinely used to interrogate key reactions of the NLRP3 inflammasome signaling cascade. Moreover, the pharmacologic tools for investigating the role of Ca2+ signaling in

inflammasome activation can affect the homeostasis of other divalent cations, for

example, Zn2+, or the activity of nonselective channels permeable to both divalent and

monovalent cations(106). In this study, we investigated the role of elevated cytosolic

[Ca2+] in NLRP3 activation in murine macrophages and dendritic cells (DC) with minimal use of pharmacologic inhibitors and by directly assaying changes in cytosolic

[Ca2+] in response to two canonical NLRP3 agonists (ATP or nigericin) that facilitate K+

efflux by mechanistically distinct reactions (see Figure 3.1 A) (107,108). Other

experiments evaluated whether changes in cytosolic [Ca2+] signaling modulate NLRP3

inflammasome activation by lysosomal destabilization. We assessed possible effects of

increased cytosolic [Ca2+] on several discrete phases of the NLRP3 inflammasome

signaling cascade: formation of ASC oligomers, processing/release of caspase-1,

processing/release of IL-1β, and kinetics of caspase-1–mediated pyroptosis. Our results

indicate that the ability of K+ efflux agonists to activate NLRP3 signaling can be

dissociated from changes in cytosolic [Ca2+]. Moreover, nigericin-stimulated increases in cytosolic [Ca2+] were temporally correlated with the onset and kinetics of pyroptosis and

were absent in DC isolated from Casp1/11−/−or Nlrp3−/− mice.

Results:

Nigericin-Induced Increases in Cytosolic [Ca2+] in LPS-Primed BMDC Occur

Downstream of the NLRP3 Inflammasome/Caspase-1/Pyroptotic Signaling Cascade

Millimolar extracellular ATP gates the opening of plasma membrane P2X7 receptor nonselective cation channels that directly facilitate both efflux of cytosolic K+

and a massive influx of extracellular Ca2+ to cause a large increase in cytosolic

[Ca2+](107). In contrast, nigericin is a lipophilic ionophore that directly functions as a

K+/H+ exchanger upon partitioning into the plasma membrane or intracellular organelles

(Figure 3.1 A). Nigericin is highly selective for monovalent cations and does not directly

facilitate transmembrane Ca2+ fluxes(108). Thus, any nigericin-induced increases in cytosolic [Ca2+] will reflect secondary changes in the activity of Ca2+ channels,

nonselective ion channels, or Ca2+ homeostatic transporters due to altered [K+] or H+ concentration within the cytosol or organellar compartments. Although ATP and nigericin trigger efflux of cytosolic K+ by distinct mechanisms, both agonists induce

similar magnitudes of caspase-1–dependent IL-1β release (Figure 3.1 B) in LPS-primed murine BMDC. The relative magnitudes of ATP-stimulated versus nigericin-stimulated

IL-1β release can vary modestly between different primary BMDC cultures, with some showing equivalent responses to the two agonists (see Figures 3.4 C, 3.5 G) others exhibiting slightly higher ATP efficacy (Figure 3.2 B; see Figure 3.5 C), and some

showing lower ATP efficacy (Figure 3.1 B). This likely reflects modest differences in the

expression levels of the ionotropic P2X7 receptors. We previously reported that accumulation of extracellular IL-1β by nigericin- and ATP-stimulated BMDC is maximal within 30 min(109). The responses are completely abrogated in Casp1/11−/− (these cells

are also deficient in caspase-11) and Nlrp3−/− BMDC (Figure 3.1 B) or in WT BMDC

Figure 3.1: Nigericin-induced increases in cytosolic [Ca2+] in LPS-primed BMDC occur downstream of the NLRP3 inflammasome/caspase-1/pyroptotic signaling cascade. (A) Diagram of changes in cation homeostasis occurring in myeloid cells in response to treatment with nigericin and ATP/P2X7. (B) IL-1β release from LPS-primed

WT, Nlrp3−/−, and Casp-1/11−/− BMDC in response to 10 μM nigericin and 5 mM ATP was measured by ELISA. Data represent mean of two independent experiments. (C) LPS-

primed WT, Nlrp3−/−, and Casp-1/11−/− BMDC were stimulated with 10 μM nigericin or

5 mM ATP for 30 min. BMDC were lysed with 10% nitric acid and lysates were

analyzed by atomic absorption spectroscopy to measure cellular [K+]. Data represent

mean of three independent experiments. (D–F) WT, Nlrp3−/−, or Casp-1/11−/− BMDC

were primed with LPS (1 μg/ml) for 4 h and loaded with 1 μg/ml fluo-4-AM for 30 min.

Baseline readings were taken for 5 min and 10 μM nigericin (NG) or 5 mM ATP were added at t = 5 min. Cytosolic [Ca2+] was determined by measuring fluo-4 fluorescence.

Data represent a mean of two independent experiments. (G–I) LPS-primed WT, Nlrp3−/−,

and Casp-1/11−/−BMDC were stimulated with 10 μM nigericin or 5 mM ATP for 30 min.

Onset of pyroptosis was determined by measuring permeability of the cell membrane to

propidium2+. Baseline readings were taken for 5 min and nigericin or ATP were added at

t = 5 min. Data represent a mean of two independent experiments.

Figure 3.1

stimulated in media with elevated extracellular [K+] to prevent efflux of cytosolic

[K+](109). Nigericin and ATP also induced robust decreases in cytosolic [K+] in WT,

Casp1/11−/−, or Nlrp3−/− cells (Figure 3.1 C). Thus, key steps in the upstream NLRP3 inflammasome signaling cascade that culminate in IL-1β release are maximally activated within this 30-min interval. To characterize how changes in cytosolic [Ca2+] are

temporally regulated during this critical phase of ATP- and nigericin-induced NLRP3

inflammasome activation, we used BMDC loaded with fluo-4 fluorescent Ca2+ sensor dye

and assayed under experimental conditions (5 × 105 cells/0.5 ml test medium/well in 24- well plates at 37°C) identical to those used to measure IL-1β release. Stimulation of LPS- primed BMDC with 5 mM ATP induced an 20-fold increase in cytosolic [Ca2+] from the basal level of 50–60 nM; the change in [Ca∼ 2+] peaked within 3 min and then modestly

decreased during the next 30 min to values 6- to 10-fold above basal (Figure 3.1 D). In contrast, cytosolic [Ca2+] did not change during the first 10–12 min after nigericin

stimulation and then gradually increased during the next 20 min to values only 3- to 4-

fold above basal (Figure 3.1 D). Notably, ATP stimulated rapid and robust increases in

cytosolic [Ca2+] in Casp1/11−/− BMDC (Figure 3.1 E) and Nlrp3−/−BMDC (Figure 3.1

F), albeit with somewhat different kinetics. In contrast, the delayed and modest increase

in cytosolic [Ca2+] triggered by nigericin in WT cells was absent in Casp1/11−/− or

Nlrp3−/− BMDC (Figures 3.1 D and 3.1 E). These findings suggest that the nigericin-

induced rise in [Ca2+] observed in WT BMDC occurs downstream of NLRP3

inflammasome activation as a result of caspase-1–dependent changes in plasma

membrane permeability.

The ability of caspase-1 to alter plasma membrane permeability as part of the

proinflammatory cell death mechanism of pyroptosis is increasingly recognized as

another readout of caspase-1 activity independent of its canonical action as an IL-1β

converting enzyme(101). Cookson and colleagues (89,110) have developed assays based

on the uptake of small cationic DNA-intercalating dyes, such as ethidium+, to track these

caspase-1–dependent changes in membrane permeability during pyroptotic progression in

macrophages infected with Salmonella or Anthrax. We reasoned that the nigericin-

induced increases in cytosolic [Ca2+] may occur as a secondary consequence of the

caspase-1–dependent increase in membrane permeability. We adapted the methods

developed by Cookson and colleagues in an assay that measured the onset and

progression of this permeability increase under the same experimental conditions used to

monitor changes in cytosolic [Ca2+] and IL-1β release. The assay tracks influx of the

2+ normally impermeant propidium organic cation (Mr of 416 Da as free cation; Mr of 668

Da as propidium iodide salt) that undergoes a large increase in 540ex→620em fluorescence

upon intercalation with nuclear DNA. Nigericin initiated an influx of propidium2+ into

WT BMDC after a 12- to 15-min delay following its addition; thereafter, the extent of

propidium2+ uptake into the BMDC population rapidly increased, with 80% of the cells

accumulating the dye during the 30-min stimulation with nigericin (Figure∼ 3.1 G). The delay phase and kinetics characterizing nigericin-induced propidium2+ influx were well correlated with the delay phase and kinetics of the nigericin-triggered increase in

cytosolic [Ca2+] (Figure 3.1 D). Although influx of propidium2+ occurred earlier (by 6–

8 min) in response to ATP than to nigericin, the time course of the ATP-stimulated ∼

propodium2+ uptake (Figure 3.1 G) was markedly different from the ATP-induced Ca2+

increase response (Figure 3.1 D). The propidium2+ influx responses to both nigericin and

ATP-gated P2X7 channels were absent in Casp1/11−/− (Figure 3.1 H) or Nlrp3−/− (Figure

3.1 I) BMDC and thus reflected induction of the pyroptotic membrane permeability transition. These studies demonstrate that the canonical NLRP3 activator nigericin does induce an increase in cytosolic [Ca2+] in murine BMDC. However, this rise in [Ca2+]

occurs downstream of inflammasome activation as a secondary consequence of the

pyroptotic membrane permeability transition (Figure 3.1 A). Enhanced Ca2+ influx may

be a general response downstream of diverse caspase-1 inflammasome platforms. Vance

and colleagues (111) reported that flagellin activation of NLRC4 inflammasomes in

tissue-resident mouse macrophages triggered a caspase-1–dependent Ca2+ influx that

preceded cytolysis and was required for phospholipase A2–dependent eicosanoid

production; this was linked to a Ca2+-dependent “eicosanoid storm” in vivo, leading to

systemic shock and death of the mice.

NLRP3 Inflammasome Signaling Responses to K+ Efflux Agonists or Lysosomal

Destabilization are Dissociated From Influx of Extracellular Ca2+

That the nigericin-induced increases in cytosolic [Ca2+] occur downstream of

NLRP3-dependent caspase-1 activation argues against a necessary role for increased

[Ca2+] in the regulation of this inflammasome pathway in response to K+ efflux stimuli in

the murine BMDC model. However, we directly tested the contribution of extracellular

Ca2+ and Ca2+ influx to nigericin- and ATP-induced NLRP3 inflammasome signaling by

2+ comparison of BMDC incubated in standard Ca -containing (1.5 mM CaCl2) BSS versus

2+ Ca -free BSS (no added CaCl2 plus supplementation with 0.3 mM EGTA). Removal of

Ca2+ from the extracellular medium eliminated the increases in cytosolic [Ca2+] triggered

by nigericin or ATP (Figure 3.2 A). Despite the absence of detectable increases in

cytosolic [Ca2+], the IL-1β release responses to nigericin or ATP were not inhibited by

the removal of extracellular Ca2+ (Figure 3.2 B and 3.2 C). Similarly, the

processing/release of caspase-1 and the formation of ASC oligomers in response to

nigericin or ATP were not attenuated when BMDC were stimulated in Ca2+-free medium

(Figure 3.2 C). Notably, the delay phases characterizing induction of the propidium2+

permeability transition in nigericin- or ATP-stimulated cells were shortened in the absence of extracellular Ca2+ and the rates of propidium2+ influx were accelerated (Figure

3.2 D). As expected, both nigericin and ATP caused significant decreases in cellular [K+]

(Figure 3.2 E), but this response was similar in either the presence or absence of

extracellular Ca2+ and measurable Ca2+ influx. These findings demonstrate that influx of

extracellular Ca2+ and the resulting increase in cytosolic [Ca2+] are not necessary signals

for NLRP3 inflammasome activation in BMDC stimulated with agents that directly

trigger rapid efflux of cytosolic K+. Brough et al. (56) reported the similar observation

that Ca2+-free medium did not inhibit, but rather increased, ATP-stimulated IL-1β release

from isolated murine peritoneal macrophages.

We also tested whether changes in cytosolic [Ca2+] are required for NLRP3 inflammasome activation by stimuli that induce lysosomal destabilization. Crystalline particulates or insoluble protein aggregates induce NLRP3 inflammasome assembly via a pathway involving phagocytosis of the particulates, phagosome maturation/fusion with lysosomes, and compromise of lysosome integrity(40,112). Because the processes of

Figure 3.2: NLRP3 inflammasome signaling responses to K+ efflux agonists are dissociated from influx of extracellular Ca2+. (A) LPS-primed BMDC were treated with 10 μM nigericin (NG) or 5 mM ATP for 30 min in the presence/absence of 1.5 mM extracellular [Ca2+]. Cytosolic [Ca2+] was determined as described in Figure 3.1. Data

represent a mean of three independent experiments. (B–E) LPS-primed BMDC were

treated for 30 min with 10 μM nigericin or 5 mM ATP in the presence/absence of 1.5

mM extracellular [Ca2+]. (B) IL-1β release was determined by ELISA. Data represent a

mean of six independent experiments. (C) Soluble lysate fraction (Lys) was probed for

procaspase-1 and pro–IL-1β, insoluble lysate pellet was crosslinked with DSS and probed

for oligomerized ASC, and extracellular medium fraction (ECM) was probed for mature

caspase-1 and IL-1β. (D) Onset of pyroptosis was determined by measuring permeability

of the cell membrane to propidium2+. Baseline readings were taken for 5 min and

nigericin or ATP was added at t = 5 min. Data represent mean of four independent

experiments. (E) BMDC were lysed with 10% nitric acid and lysates were analyzed by

atomic absorption spectroscopy to measure cellular [K+]. Data represent mean of three

independent experiments.

Figure 3.2

particulate phagocytosis and phagolysosomal maturation per se may be modulated by

perturbation of Ca2+ signaling, we used the soluble lysosomotropic agent LLME to

induce rapid and synchronous lysosome disruption in LPS-primed BMDC. LLME enters

cells via amino acid transporters, accumulates in lysosomes, and is then converted into

membrane-disruptive polyleucine peptides (e.g., Leu4, Leu6) via a reverse condensation reaction catalyzed by the cathepsin C/dipeptidyl peptidase I(113). It has been widely used as an NLRP3 activation stimulus in multiple inflammatory models(53). A 30-min incubation with 1 mM LLME induced robust release of IL-1β from WT, but not

Casp1/11−/− or Nlrp3−/− BMDC (Figure 3.3 A). Consistent with previous findings in murine macrophages(53), LLME stimulated equivalent K+ efflux from WT, Casp1/11−/−,

and Nlrp3−/− BMDC (Figure 3.3 B). Although the mechanism by which LLME or

particulate lysososomal destabilizing stimuli elicit K+ efflux has not been defined, Zhong

et al. (62) reported that aluminum hydroxide and silica induce activation of TRMP2

nonselective cation channels in murine macrophages as assayed by Ca2+ influx; there was

also partial inhibition of IL-lβ release in Trpm2−/− cells. Notably, LLME induced Ca2+

influx into BMDC after a 5-min lag period to result in a >10-fold sustained increase in

cytosolic [Ca2+] within 20 min (Figure 3.3 D). This increase was absent when BMDC

were stimulated by LLME in Ca2+-free BSS. Despite the absence of a detectable increase in cytosolic [Ca2+] under these latter conditions, the IL-1β release response to LLME was not inhibited, but rather was potentiated, by removal of extracellular Ca2+ (Figure 3.3 C).

LLME also stimulated propidium2+ influx after a 15-min lag period under control (1.5

mM extracellular Ca2+) conditions (Figure 3.3 E); removal of extracellular Ca2+ modestly

shortened the lag phase and increased the rate of propidium2+ influx. Thus, lysosome

Figure 3.3: NLRP3 inflammasome signaling responses to lysosomal destabilization

are dissociated from influx of extracellular Ca2+. (A–C) LPS-primed BMDC were stimulated with 1 mM LLME for 30 min. (A) LPS-primed WT, Nlrp3−/−, and Casp1−/−

BMDC were stimulated with LLME and IL-1β release was determined by ELISA. Data

represent a mean of two independent experiments. (B) LPS-primed WT, Nlrp3−/−, and

Casp1−/− BMDC were stimulated with LLME, lysed with 10% nitric acid, and the lysates

were analyzed by atomic absorption spectroscopy to measure cellular [K+]. Data

represent mean of three independent experiments. (C) LPS-primed WT BMDC were stimulated with LLME in the presence/absence of 1.5 mM extracellular [Ca2+] and IL-1β

release was determined by ELISA. Data represent a mean of two independent

experiments. (D and F) Cytosolic [Ca2+] was determined as described in Figure 3.1 in

WT (D and F), Nlrp3−/−, and Casp1−/− (F) BMDC. Baseline readings were taken for 5 min

and LLME was added at t = 5 min. Data represent a mean of two independent

experiments. (E) LLME-induced propidium2+ influx was measured in LPS-primed

BMDC in the presence/absence of 1.5 mM extracellular [Ca2+]. Baseline readings were

taken for 5 min and LLME was added at t = 5 min. Data represent mean of two

independent experiments.

Figure 3.3

destabilization, similar to ATP gating of P2X7 channels, induces a Ca2+ influx that

precedes, but is not required for, caspase-1–dependent IL-1β release or the propidium2+

influx response, which tracks with caspase-1 activation. Notably, the rates of LLME-

stimulated Ca2+ influx were attenuated in Casp1/11−/− and Nlrp3−/− BMDC (Figure 3.3

F). This suggests that the net Ca2+ influx response induced by LLME in WT DC reflects contributions from both an inflammasome-independent cation permeability pathway (that also mediates K+ efflux) and an inflammasome/caspase-1–dependent change in

permeability (that also mediates propidium2+ influx).

NLRP3 Inflammasome Signaling Responses to K+ Efflux Agonists are Dissociated From

Release of Thapsigargin-Sensitive Intracellular Ca2+ Stores

The absence of extracellular Ca2+ effectively eliminated the increases in cytosolic

[Ca2+] in response to nigericin or ATP but did not attenuate NLRP3 inflammasome activation in BMDC. However, several reports have suggested that mobilization of intracellular Ca2+ stores may be the critical regulatory signal. These studies have

proposed that microdomains of the ER membrane system adjacent to mitochondria can

release sufficient local Ca2+ to induce mitochondrial dysfunction leading to generation of

reactive oxygen species and release of mitochondrial DNA into the cytosol. The

mitochondria-derived reactive oxygen species and DNA then directly activate NLRP3

conformational changes to drive inflammasome assembly and signaling(114). To investigate the role of released ER Ca2+ stores in the activation of NLRP3 inflammasome signaling, we treated BMDC with thapsigargin, an inhibitor of the ER Ca2+ ATPase, for

30 min prior to stimulation with nigericin or ATP. Submicromolar thapsigargin inhibits

all isoforms of the sarcoplasmic/ER calcium ATPases that actively maintain high

intraluminal concentrations (0.3–1 mM) of free Ca2+ within the ER(115). Thapsigargin

inhibition of sarcoplasmic/ER calcium ATPase pump activity facilitates the rapid efflux

of this stored Ca2+ into the cytosol via as yet undefined “leak” channels. Ca2+ released

into the cytosol is rapidly transported to the extracellular compartment via the combined

actions of the plasma membrane Ca2+ ATPase pump and Na+/Ca2+ exchange transporters.

However, the reduction in intraluminal [Ca2+] in thapsigargin-treated cells also induces

oligomerization of STIM family sensor proteins in the ER membrane, and the

oligomerized STIM puncta activate conformational changes in Orai family store-operated

Ca2+ influx channels within juxtaposed domains of the plasma membrane(116). In the

presence of extracellular Ca2+, this STIM-dependent gating of Orai channels facilitates

Ca2+ influx to offset the loss of intracellular Ca2+ via plasma membrane Ca2+ ATPase and

Na+/Ca2+ exchange activity and thereby replenishes and sustains the intracellular Ca2+

pools within the ER. Thus, we routinely treated BMDC with thapsigargin in Ca2+-free

BSS (as in Figures 3.2 and 3.3) to eliminate Ca2+ influx via activated STIM/Orai

complexes and any replenishment of the ER Ca2+ stores(116). As expected, stimulation of thapsigargin-treated BMDC with nigericin or ATP in the absence of extracellular Ca2+

abolished the increases in cytosolic [Ca2+] induced by these NLRP3 activators (Figure 3.4

A, open symbols). However, treatment of BMDC with thapsigargin under such Ca2+-free

conditions did not delay the onset of pyroptotic membrane permeability transition or

inhibit IL-1β release in response to nigercin or ATP (Figures 3.4 B and 3.4 C). Positive

control experiments using cells treated with thapsigargin in Ca2+-containing BSS verified

Figure 3.4: NLRP3 inflammasome signaling responses to K+ efflux agonists are dissociated from release of thapsigargin-sensitive intracellular Ca2+ stores. (A) LPS-

primed BMDC were treated with 300 nM thapsigargin (TG) at t = 0 in the absence of

extracellular Ca2+. BMDC were washed at t = 30 min and 10 min of baseline readings

were taken prior to addition of 10 μM nigericin or 5 mM ATP at t = 40 min. Cytosolic

[Ca2+] was determined as described in Figure 3.1. Data represent a mean of three

independent experiments. (B and C) LPS-primed BMDC were treated with 300 nM

thapsigargin in the absence of extracellular Ca2+. BMDC were then stimulated with 10

μM nigericin or 5 mM ATP in the absence of extracellular Ca2+. (B) Onset of pyroptosis

was determined by measuring permeability of the cell membrane to propidium2+.

Baseline readings were taken for 5 min and nigericin or ATP was added at t = 5 min.

Data represent a mean of three independent experiments. (C) IL-1β release was

determined by ELISA. Data represent a mean of six independent experiments.

the efficacy of thapsigargin as a Ca2+-mobilizing agent in this BMDC model system

(Figure 3.4 A, closed symbols). These findings demonstrate that release of ER Ca2+ stores

is also not a necessary signal for NLRP3 inflammasome activation in BMDC simulated

with K+ efflux agonists. Previously, Menu et al. (117) observed that micromolar

concentrations of thapsigargin activate NLRP3 inflammasome signaling via induction of

an ER stress response. In the above experiments we used a lower concentration of

thapsigargin (300 nM) than in the Menu et al. study and did not observe induction of

caspase-1–dependent pyroptosis or significant IL-1β release by BMDC in response to

treatment with thapsigargin alone (Figures 3.4 B and 3.4 C).

NLRP3 Inflammasome Signaling Responses to K+ Efflux Agonists are Dissociated From

Changes in Cytosolic [Ca2+] in BMDC Primed With TLR2 or TNF Receptor Agonists

Although the above findings indicate that increases in cytosolic [Ca2+] are not

required for nigericin- or ATP-stimulated NLRP3 activation in BMDC, it is possible that

Ca2+ may act as a modulatory signal by increasing the efficiency of productive NLRP3

inflammasome assembly under conditions of submaximal signal 1 priming and/or signal

2 activation. In all experiments described thus far, LPS was used as the priming stimulus

to upregulate expression of NLRP3 and pro–IL-1β. Given the ability of TLR4 to drive

both MyD88- and TRIF-dependent signaling pathways, LPS is a particularly strong

activator of the NF-κB–based transcription of these proinflammatory gene products, and

we have previously reported that NLRP3 is expressed at high levels in LPS-primed

BMDC(109). With high levels of expression of inflammasome components, potential

modulatory effects of elevated cytosolic [Ca2+] on NLRP3 activation or inflammasome

complex assembly may be negligible during stimulation with potent signal 2 agonists

such as ATP or nigericin. To test for more subtle modulatory effects of cytosolic [Ca2+]

on NLRP3 inflammasome activation, we used less efficacious (relative to LPS) signal 1

priming stimuli, including the synthetic TLR2 agonist Pam3CSK4 and the

proinflammatory cytokine TNF-α. Both nigericin and ATP triggered increases in

2+ cytosolic [Ca ] in BMDC primed with Pam3CSK4 (Figure 3.5 A) or TNF-α (Figure 3.5

E) that were similar in kinetics to those observed in the LPS-primed cells (Figure 3.1 D).

Control experiments verified that, as expected, nigericin induced similar magnitudes of

+ K efflux in Pam3CSK4- and TNF-α–primed cells as in unprimed or LPS-primed BMDC.

Importantly, the nigericin-induced increases in Ca2+ influx occurred after a 10- to 15-min

delay and correlated with the onset of pyroptotic propidium2+ influx in both the

Pam3CSK4-primed (Figure 3.5 B) and TNF-α–primed BMDC (Figure 3.5 F). Consistent

with the reduced efficacy of these signal 1 stimuli, the magnitudes of nigericin- or ATP-

stimulated IL-1β production in Pam3CSK4 -primed cells (Figure 3.5 C: 12–20 ng/ml/30 min in Ca2+-containing saline), and especially in TNF-α–primed cells (Figure∼ 3.5 G: 1.5

ng/ml/30 min), were lower than in LPS-primed cells (Figures 3.1 B, 3.2 B, 3.3 C: 15–∼25

ng/ml/30 min). Similarly, the percentages of BMDC that accumulated proprodium2+ after

30 min of nigericin or ATP stimulation in Ca2+-containing saline were lower with

Pam3CSK4 priming (Figure 3.5 B: 50% with both agonists) and TNF-α priming (Figure

3.5 F: 45% with nigericin and 15% with ATP) than with LPS priming (Figures 3.1 G, 3.2

D: 60–80% with both agonists). The absence of extracellular Ca2+ did not inhibit the

pyroptotic propidium2+ influx (Figures 3.5 B, 3.5 F) or IL-1β release (Figures 3.5 C, 3.5

Figure 3.5: NLRP3 inflammasome signaling responses to K+ efflux agonists are dissociated from changes in cytosolic [Ca2+] in BMDC primed with TLR2 or TNF

receptor agonists. (A and E) BMDC were primed with Pam3CSK4 (2 μg/ml) or TNF-α

(100 ng/ml) for 4 h. Baseline fluorescence measurements were taken for 5 min prior to

addition of 10 μM nigericin or 5 mM ATP at t = 5 min. Cytosolic [Ca2+] was determined as described in Figure 3.1. Data represent a mean of two independent experiments. (B, C,

F, and G) Pam3CSK4-primed or TNF-α–primed BMDC were treated with 10 μM

nigericin or 5 mM ATP for 30 min in the presence or absence of 1.5 mM extracellular

[Ca2+]. (B and F) Onset of pyroptosis was determined by measuring permeability of the cell membrane to propidium2+. Baseline readings were taken for 5 min and nigericin or

ATP was added at t = 5 min. Data represent mean of two independent experiments. (C

and G) IL-1β release from Pam3CSK4-primed BMDC or TNF-α–primed BMDC was

determined by ELISA. Data represent a mean of two independent experiments for each

panel. (D and H) Pam3CSK4-primed or TNF-α–primed BMDC were pretreated with 300

nM thapsigargin (TG) for 30 min in the absence of extracellular Ca2+. BMDC were

washed and stimulated with 10 μM nigericin or 5 mM ATP for 30 min in the absence of

2+ extracellular Ca . IL-1β release from Pam3CSK4-primed BMDC or TNF-α–primed

BMDC was determined by ELISA. Data represent a mean of two independent

experiments for each panel.

Figure 3.5

G) responses to nigericin or ATP in BMDC primed with Pam3CSK4 or TNF-α. Rather, as

2+ observed in LPS-primed BMDC, removal of extracellular Ca from the Pam3CSK4- or

TNF-α–primed BMDC during stimulation shortened the delay phases and increased the

rates of the propidium2+ uptake responses. In Ca2+-free medium, there was a trend (that

did not reach statistical significance) for ATP, but not nigericin, to stimulate more IL-1β

release in the TNF-α–primed cells (Figure 3.5 G). Interestingly, thapsigargin treatment

(in Ca2+-free saline) produced a modest (20–33%) decrease in the IL-1β release responses

to ATP, but not nigericin, in the Pam3CSK4- and TNF-α–primed BMDC (Figure 3.5 D,

3.5 H). Taken together, the experiments with Pam3CSK4- or TNF-α–primed BMDC also argue against a necessary role for increased Ca2+ influx and/or mobilization of

intracellular Ca2+ stores in the activation of NLRP3 inflammasome signaling by K+ efflux

agonists.

NLRP3 Inflammasome Signaling Responses to K+ Efflux are Dissociated From Changes

in Cytosolic [Ca2+] in Murine Macrophages

Most studies implicating roles for increased cytosolic [Ca2+] in activation of

NLRP3 inflammasome signaling have been performed in murine or human

macrophages(56-59,104). Although the key elements of NLRP3 inflammasome

composition and function are conserved in macrophage and DC models, some differences

in modulatory signals have been reported. For example, phosphorylation of ASC by Syk

markedly potentiates NLRP3 inflammasome activation by nigericin in BMDM and

peritoneal macrophages, but not BMDC(74). Therefore, we investigated the potential

contribution of extracellular Ca2+ and ER Ca2+ stores to NLRP3 inflammasome activation

in BMDM. As in BMDC, nigericin and ATP induced increases in cytosolic [Ca2+] with

distinctive time courses in BMDM (Figure 3.6 A). ATP triggered a rapid 20-fold increase

from the 50 nM basal [Ca2+] in the macrophages that peaked at 5 min poststimulation.

However, the subsequent decrease was more rapid and robust than that observed in the

BMDC (Figure 3.1 D). As in DC, the ATP-induced Ca2+ influx preceded the induction of

propidium2+ influx by 8 min in the BMDM (Figure 3.6 B). The nigericin-induced

increase in macrophage∼ [Ca2+] was defined by a similar 10- to 12-min delay phase

(Figure 3.6 A) as observed in the DC (Figure 3.1 D). This was followed by a slow rate of increase that plateaued at 200–300 nM (Figure 3.6 A); the delayed increase in [Ca2+] was

also correlated with the onset of pyroptotic propidium2+ influx in the BMDM (Figure 3.6

B). As in BMDC, removal of extracellular Ca2+ did not suppress either the kinetics or the

magnitudes of the propidium2+ influx responses to nigericin and ATP in BMDM (Figure

3.6 B). Similarly, stimulation of macrophages with nigericin or ATP in Ca2+-free saline did not inhibit, but rather had no effect (nigericin) or modestly enhanced (ATP), the IL-

1β release responses (Figure 3.6 C). The combined removal of extracellular Ca2+ and

depletion of ER Ca2+ stores by thapsigargin treatment in BMDM did not significantly change IL-1β release in response to either agonist (Figure 3.6 D). These findings indicate that increased cytosolic [Ca2+] is a not a necessary signal for NLRP3 inflammasome

activation in murine BMDM.

Figure 3.6: NLRP3 inflammasome signaling responses to K+ efflux agonists are dissociated from changes in cytosolic [Ca2+] in murine macrophages. (A) Murine

BMDM were primed with LPS (1 μg/ml) for 4 h. Baseline readings were taken for 5 min

and 10 μM nigericin (NG) or 5 mM ATP was added at t = 5 min. Cytosolic [Ca2+] was

determined as described in Figure 3.1. Data represent a mean of two independent

experiments. (B and C) LPS-primed BMDM were treated for 30 min with 10 μM nigericin or 5 mM ATP in the presence/absence of 1.5 mM extracellular [Ca2+]. (B)

Onset of pyroptosis was determined by measuring permeability of the cell membrane to

propidium2+. Baseline readings were taken for 5 min and nigericin or ATP was added at t

= 5 min. Data represent mean of four independent experiments. (C) IL-1β release was

determined by ELISA. Data represent a mean of four independent experiments. (D) LPS-

primed BMDM were treated with 300 nM thapsigargin for 30 min in the absence of

extracellular Ca2+ and then stimulated with 10 μM nigericin or 5 mM ATP for 30 min in

the absence of extracellular Ca2+. IL-1β release was determined by ELISA. Data

represent a mean of four independent experiments.

Figure 3.6

Increased Cytosolic [Ca2+] Induced by Ca2+ Ionophore or Ca2+-Mobilizing GPCR is not a Sufficient Signal for NLRP3 Inflammasome Activation

Several studies have reported that stimulation of myeloid cells with agonists for

certain Gq- or Gi-coupled receptors induces NLRP3 inflammasome activation via

phospholipase C (PLC)–mediated generation of inositol trisphosphate (IP3) and release of

ER Ca2+ stores(58,59,105). We compared the efficacy of various Ca2+-mobilizing agonists versus the K+ efflux agonists in the activation of NLRP3 inflammasome

signaling by stimulating LPS-primed BMDC under the same test conditions (30 min in

1.5 mM CaCl2-containing saline) routinely used for induction by nigericin or P2X7

channel gating. The Ca2+-mobilizing agonists included 1) ionomycin, a Ca2+ ionophore

that stimulates both influx of extracellular Ca2+ and release of ER Ca2+ stores(118); 2)

R568, a synthetic agonist of the Gq-coupled CaSR reported to induce robust NLRP3 inflammasome activation in murine and human macrophages via the PLC/IP3-gated ER

Ca2+ mobilization pathway(58,59); 3) ATP or UTP, which at submillimolar

concentrations activate the Gq/PLC-coupled P2Y2 nucleotide receptors highly expressed in all myeloid leukocyte subtypes(119); and 4) fMLF, a synthetic agonist of the Gi/PLC-

coupled formyl peptide receptors (FPR) that facilitate chemoattraction of myeloid

leukocytes to local accumulations of bacteria-derived formylated proteins(120).

As expected, ionomycin mimicked the ability of ATP-gated P2X7 channels to induce a large and immediate increase in cytosolic [Ca2+] that was sustained for up to 30

min (Figure 3.7 A). Despite this robust elevation in cytosolic [Ca2+], the 30-min treatment

with ionomycin did not elicit statistically significant release of IL-1β as indicated by

ELISA (Figure 3.7 B) or Western blot for the mature 17-kDa cytokine (Figure 3.7 G).

These observations in BMDC are consistent with Brough et al. (56) and Murakami et al.

(57) who reported a similar inability of ionomycin to activate caspase-1 and release of

mature IL-1β in murine macrophages. Ionomycin also did not trigger rapid pyroptotic signaling in the BMDC as demonstrated by the lack of propidium2+ influx throughout the ionomycin exposure (Figure 3.7 E). Ionomycin did induce a modest 25–30% reduction in cellular K+ content (Figure 3.7 F) that was independent of NLRP3 or caspase-1

expression (Figure 3.8 A). Brough et al. (56) described an ionomycin-induced decrease in

cell viability (release of 64% total LDH) in murine macrophages, which would

necessarily be correlated with a decrease in total K+ content. We observed much less

ionomycin-induced cell death (release of 18–20% total LDH within 60 min) in either WT

or Casp1/11−/− BMDC (Figure 3.8 B), which contrasted with the 70% LDH release in

ATP-treated WT cells and no LDH release in ATP-treated Casp1/11−/− cells. Thus, the

25–30% decrease in [K+] induced by ionomycin reflects both cell death–dependent and cell death–independent components. The magnitude of this [K+] decrease was significantly smaller than that triggered by nigericin or P2X7 channel gating (Figure 3.7

F). Notably, this decrease was not sufficient for robust inflammasome assembly as indicated by the barely detectable accumulation of ASC oligomers (Figure 3.7 G). The findings further suggest that cytosolic [K+] must decrease below a threshold value to

entrain the signaling pathways required for conformational activation of NLRP3.

All of the tested GPCR agonists triggered an immediate 4-fold increase in

cytosolic [Ca2+] that peaked within 60 s and then rapidly decreased during the next few

minutes (Figure 3.7 C). The fMLF-, UTP-, or 100 μM ATP-triggered Ca2+ transients

Figure 3.7: Increased cytosolic [Ca2+] induced by Ca2+ ionophore or Ca2+-mobilizing

GPCR is not a sufficient signal for NLRP3 inflammasome activation. (A) Cytosolic

[Ca2+] was determined as described in Figure 3.1. Baseline readings were taken for 5 min

and 5 mM ATP or 3 μM ionomycin was added at t = 5 min. Data represent a mean of two independent experiments. (B) LPS-primed BMDC were treated with 10 μM nigericin

(NG), 5 mM ATP, or 3 μM ionomycin for 30 min. IL-1β release was measured by

ELISA. Data represent the mean of 10 independent experiments. (C) LPS-primed BMDC were treated with 30 μM R568, 0.1 mM ATP, 1 μM fMLF, or 0.1 mM UTP for 30 min.

Cytosolic calcium levels were determined as described in Figure 3.1. Baseline readings were taken for 5 min and GPCR agonists were added at t = 5 min. Data are representative of two independent experiments. (D) LPS-primed BMDC were treated with calcium- mobilizing agents for 30 min. IL-1β release was measured by ELISA. Data represent mean of five independent experiments. (E) LPS-primed BMDC were treated with 10 μM nigericin, 5 mM ATP, or 3 μM ionomycin. Onset of pyroptosis was determined by measuring permeability of the cell membrane to propidium2+. Baseline readings were taken for 5 min and nigericin, ATP, or ionomycin was added at t = 5 min. Data represent a mean of two independent experiments. (F) LPS-primed BMDC were treated with 10

μM nigericin, 5 mM ATP, or 3 μM ionomycin. Cells were lysed with 10% nitric acid and lysates were analyzed by atomic absorption spectroscopy to measure cellular [K+]. Data

represent mean of eight independent experiments. (G) LPS-primed BMDC were

stimulated with 10 μM nigericin, 5 mM ATP, 3 μM ionomycin, or 30 μM R568 for 30

min. Soluble lysate fraction (Lys) was probed for procaspase-1 and pro–IL-1β, insoluble

76 lysate pellet was crosslinked with DSS and probed for oligomerized ASC, and extracellular medium fraction (ECM) was probed for mature caspase-1 and IL-1β.

Figure 3.7

Figure 3.8: Effects of ionomycin on K+ efflux and cell death in wildtype, Casp1/11-/-, or Nlrp3-/- BMDC. (A) LPS-primed wildtype, Casp1/11-/-, or Nlrp3-/- BMDC were treated with 3μM ionomycin for 30 min. Cells were lysed with 10% nitric acid and lysates were analyzed by atomic absorption spectroscopy to measure cellular [K+]. Data represent mean of triplicate determinations from a single experiment. (B) LPS-primed wildtype or Casp1/11-/-BMDC were treated with 5 mM ATP or 3μM ionomycin for the indicated times prior to collection of the extracellular medium for analysis of LDH release. The LDH release was normalized to the total LDH content measured in triton lysates. Data represent mean of triplicate determinations from a single experiment.

decayed to the basal level within 5–7 min. In contrast, the R568-induced peak in cytosolic [Ca2+] was followed by a sustained 3-fold elevation in cytosolic [Ca2+] during

the 30-min test period (Figure 3.7 C). No statistically significant release of IL-1β was

observed in BMDC stimulated for 30 min with fMLF, UTP, 100 μM ATP, or R568

(Figure 3.7 D). Similarly, R568 did not induce detectable accumulation of mature IL-1β or caspase-1 p20 subunits in the extracellular medium (Figure 3.7 G) and elicited only weak accumulation of intracellular ASC oligomers (Figure 3.7 G). These data demonstrate that agonists for Ca2+-mobilizing GPCR do not mimic the ability of K+

efflux agonists to elicit rapid and robust NLRP3 inflammasome activation in the murine

BMDC model.

Suppression of Nigericin-Stimulated NLRP3 Inflammasome Signaling by BAPTA and 2-

APB can be Dissociated from Perturbation of Ca2+ Signaling

Some support implicating Ca2+ signaling in NLRP3 inflammasome activation is

based on observations that BAPTA, a strong Ca2+ chelator and buffer of cytosolic Ca2+,

2+ and 2-APB, an inhibitor of IP3-gated Ca release channels and store-operated calcium

entry channels, strongly suppress IL-1β release in response to canonical NLRP3 activators(56-58,104). Given our finding that removal of extracellular Ca2+ (with or

without thapsigargin treatment) eliminates the increases in cytosolic [Ca2+], but not the

IL-1β release, stimulated by nigericin or ATP (Figures 3.2 and 3.4), it is possible that the

inhibitory actions of BAPTA and 2-APB on NLRP3 signaling may also be dissociated

from effects on Ca2+ signaling. We observed that loading LPS-primed BMDC with

BAPTA markedly attenuated multiple readouts of nigericin-stimulated NLRP3

inflammasome signaling, including total IL-1β release (Figure 3.9 A), extracellular

accumulation of p20 caspase-1 subunit and p17 mature IL-1β (Figure 3.9 D), formation of ASC oligomers (Figure 3.9 D), and induction of pyroptotic propidium2+ influx (Figure

3.9 C). Importantly, note that all assays were performed in the absence of extracellular

Ca2+, which effectively eliminates any nigericin-induced increase in cytosolic [Ca2+]

(Figure 3.2 A). In contrast, the nigericin-stimulated efflux of cytosolic K+ was not

inhibited in the BAPTA-loaded cells (Figure 3.9 B). We verified the efficacy of BAPTA

loading and its ability to buffer the delayed increase in cytosolic [Ca2+] induced by nigericin stimulation when BMDC were incubated in 1.5 mM Ca2+-containing basal

saline (Figure 3.10). These findings demonstrate that cytosolic BAPTA loading can

inhibit IL-1β processing and release independently of its function as a Ca2+ chelator and

downstream of the necessary NLRP3-activating K+ efflux signal.

Previous studies have demonstrated that 2-APB strongly inhibits NLRP3

inflammasome activation in response to nigericin and other stimuli. This suppression has

been ascribed to the extensively characterized actions of this reagent as an inhibitor of

2+ 2+ both IP3-gated Ca release channels in the ER and store-operated Ca influx channels in the plasma membrane(57,58). An expectation of this proposed mechanism is that the ability of 2-APB to inhibit nigericin-stimulated IL-1β release and pyroptosis should be highly correlated with suppression of Ca2+ mobilization and influx. We tested the effects of 2-APB on multiple indices of nigericin-activated NLRP3 inflammasome signaling in

LPS-primed BMDC incubated in CaCl2-containing medium. Consistent with previous

findings(57,58), we observed that 2-APB completely suppressed total IL-1β release

Figure 3.9: Suppression of nigericin-stimulated NLRP3 inflammasome signaling by

BAPTA can be dissociated from perturbation of Ca2+ signaling. LPS-primed BMDC

were loaded with 25 μM BAPTA-AM for 45 min per the manufacturer’s protocol. Cells

were then treated with 10 μM nigericin for 30 min in the absence of extracellular Ca2+.

(A) IL-1β release was measured by ELISA. Data represent a mean of three independent

experiments. (B) Efflux of cellular K+ was measured by atomic absorption spectroscopy.

Data represent a mean of three independent experiments. (C) Onset of pyroptosis was determined by measuring permeability of the cell membrane to propidium2+. Baseline

readings were taken for 5 min and 10 μM nigericin was added at t = 5 min. Data

represent a mean of four independent experiments. (D) LPS-primed BMDC were stimulated with 10 μM nigericin for 30 min in the presence/absence of BAPTA loading.

Soluble lysate fraction (Lys) was probed for procaspase-1 and pro–IL-1β, insoluble lysate pellet was crosslinked with DSS and probed for oligomerized ASC, and extracellular medium fraction (ECM) was probed for mature caspase-1 and IL-1β.

Figure 3.9

Figure 3.10: Effects of BAPTA on nigericin-induced increases in cytosolic [Ca2+] in

BMDC incubated in Ca2+-containing media. LPS-primed BMDC were loaded with

1μg/ml fluo-4 AM +/- 25μM BAPTA-AM for 45 min. Each well was briefly washed with

PBS prior to the addition of 0.5 ml 1.5mM CaCl2-containing BSS supplemented with

2.5mM probenecid. Baseline fluorescence (485nm excitation → 528 nm emission) was recorded for 10 min at 30 sec intervals. BMDC in each well were then stimulated by addition of 10μM nigericin or no agonist (arrow) and the fluorescence recorded for 50 min at 30 sec intervals. Assays were terminated by permeabilization of the cells with 1% triton X-100 for maximum Ca2+-dependent fluorescence followed by 15mM

EGTA/50mM Tris to chelate Ca2+ for minimum Ca2+-independent fluorescence. Y-axis shows the changes in relative fluorescence units (RFU). Data are representative of results from three similar experiments.

(Figure 3.11 A), extracellular accumulation of p20 caspase-1 subunit and p17 mature IL-

1β (Figure 3.11 C), formation of ASC oligomers (Figure 3.11 C), and induction of

propidium2+ influx (Figure 3.11 D) in response to nigericin. In contrast, robust K+ efflux

responses were observed in nigericin-treated BMDC in the absence or presence of 2-APB

(Figure 3.11 B). Despite the complete suppression of these multiple readouts of NLRP3 inflammasome activity, 2-APB did not suppress, but rather strongly potentiated, the nigericin-induced increase in cytosolic [Ca2+] (Figure 3.11 E). Moreover, the initial

addition of 2-APB to LPS-primed BMDC elicited an immediate 10- to 20-fold increase in

cytosolic [Ca2+] followed by a decay to a level 5-fold above basal (Figure 3.11 E), which,

even in the absence of a subsequent nigericin stimulus, was steadily maintained for many

minutes. These findings demonstrate that 2-APB inhibits NLRP3 inflammasome

signaling downstream of K+ efflux signals and independently of its canonical actions as

2+ an inhibitor of IP3 receptor/store-operated calcium entry–mediated increases in [Ca ]. In

contrast to a presumed causal relationship between its inhibitory effects on NLRP3

signaling and the suppression of Ca2+ signaling, 2-APB by itself triggered large increases in cytosolic [Ca2+] and potentiated nigericin-induced Ca2+ influx in LPS-primed murine

DC.

Figure 3.11: Suppression of nigericin-stimulated NLRP3 inflammasome signaling by

2-APB can be dissociated from perturbation of Ca2+ signaling. (A–C) LPS-primed

BMDC were incubated with 100 μM 2-APB for 20 min prior to treatment with 10 μM

nigericin for 30 min in the presence of 1.5 mM extracellular [Ca2+]. (A) IL-1β release

was measured by ELISA. Data represent a mean of three independent experiments. (B)

Efflux of cellular K+ was measured by atomic absorption spectroscopy. Data represent a mean of three independent experiments. (C) Soluble lysate fraction (Lys) was probed for procaspase-1 and pro–IL-1β, insoluble lysate pellet was crosslinked with DSS and probed for oligomerized ASC, and extracellular medium fraction (ECM) was probed for mature caspase-1 and IL-1β. (D) LPS-primed BMDC were preincubated with 100 μM 2-APB for

20 min in the presence of 1.5 mM extracellular [Ca2+] and onset of pyroptosis in response to nigericin was assayed by measuring permeability of cells to propidium2+. Baseline

readings were taken for 5 min and 10 μM nigericin was added at t = 5 min. (E) Cytosolic

[Ca2+] in LPS-primed BMDC was measured using fluo-4 fluorescence in the presence of

1.5 mM extracellular [Ca2+]. Baseline readings were taken for 5 min. Cells were treated

with 100 μM 2-APB at t = 5 min (double arrow) and 10 μM nigericin (single arrow) at t =

15 min. Data are representative of two independent experiments.

Figure 3.11

Discussion:

Perturbation of intracellular ion homeostasis is a major cellular stress signal for

activation of NLRP3 inflammasome signaling. However, the relative contributions of

decreased cytosolic [K+] versus increased cytosolic [Ca2+] remain disputed and

incompletely defined. This study provides three major findings relevant to this

unresolved area of NLRP3 regulation. First, increased cytosolic [Ca2+] is neither a

necessary nor sufficient signal for the NLRP3 inflammasome cascade induced in murine

DC and macrophages during activation by endogenous ATP-gated P2X7 receptor channels, the bacterial ionophore nigericin, or LLME-induced lysosomal disruption.

These stimuli are widely used as highly efficacious inducers of NLPR3 inflammasome assembly in murine and human myeloid leukocyte models. Second, agonists for three

Ca2+-mobilizing GPCR expressed in murine myeloid leukocytes (FPR, P2Y2 purinergic

receptor, CaSR) were ineffective as robust activators of NLRP3 signaling when directly

compared with the K+ efflux agonists under identical experimental conditions. Third,

BAPTA and 2-APB, widely used reagents for disruption of Ca2+-dependent signaling pathways, strongly suppress nigericin-induced NLRP3 inflammasome signaling via mechanisms dissociated from their canonical or expected effects on Ca2+ homeostasis.

We assessed the possible roles for influx of extracellular Ca2+ and/or mobilization

of ER Ca2+ stores on multiple steps in the serial NLRP3 signaling pathway, including

efflux of cytosolic K+, accumulation of stable ASC oligomers, production of active

caspase-1, processing and release of mature IL-1β, and induction of pyroptotic changes in

plasma membrane permeability. Direct measurements using cells loaded with fluo-4 Ca2+

sensor dye verified the absence of cytosolic [Ca2+] increases during manipulations

designed to eliminate Ca2+ influx and mobilization in the ATP- or nigericin-treated cells.

Recent reports have indicated that NLRP3 activation triggers the rapid assembly of prion-

like ASC aggregates that comprise the critical and essentially irreversible step in coupling

conformational changes in the NLRP3 stress sensor to activation of the caspase-1 effector

enzyme(37,38). Our findings indicate that ATP and nigericin induce equivalent

accumulation of detergent-insoluble ASC aggregates regardless of the presence or

absence of increases in cytosolic [Ca2+].

Although our data indicate that Ca2+ is not a critical second messenger for the

very proximal steps of the NLRP3 signaling cascade, changes in cytosolic [Ca2+] may

modulate reactions downstream of the NLRP3-dependent assembly of the ASC oligomeric platforms for caspase-1 activation, particularly the several nonclassical export pathways for release of mature IL-1β and caspase-1 itself. We previously reported that increases in cytosolic [Ca2+] do not affect caspase-1–dependent processing of pro–IL-1β

but can potentiate the release of processed mature IL-1β in some, but not all, cell

models(86,121). Depending on cell type and activation stimulus, IL-1β can be released in

different proportions via three vesicular and one nonvesicular mechanism(82). The

vesicular mechanisms include 1) IL-1β trapped within plasma membrane-derived

microvesicles that bleb and scission from the cell surface, 2) IL-1β packaged within

exosomes contained in multivesicular endosomes that subsequently fuse with the cell

surface membrane, and 3) exocytosis of IL-1β that has been internalized within secretory autophagolysosomes. The nonvesicular pathway is via regulated cell lysis as a consequence of caspase-1–driven pyroptosis. In our LPS-primed BMDC and BMDM

models, the magnitudes of IL-1β release induced by nigericin or ATP were largely

equivalent in the absence or presence of extracellular Ca2+ or in the absence or presence of thapsigargin treatment to deplete ER Ca2+ stores. We also measured propidium2+ influx

as an index of the caspase-1–regulated transition in plasma membrane permeability that

accompanies pyroptotic cell lysis(90). This parameter provides a readout of caspase-1

activation kinetics independent of IL-1β release by the vesicular mechanisms. Notably,

DC and macrophages stimulated with nigericin or ATP in the absence or presence of extracellular Ca2+ exhibited similar onset and rates of propidium2+ influx. This indicates

that the Ca2+-independent pathway predominantly mediates the nonclassical export of IL-

1β following rapid inflammasome activation by K+ efflux agonists. However, in other inflammasome models with slower progression to pyroptosis, Ca2+-dependent vesicular

IL-1β release may be more important. Differential roles for local mobilization of ER Ca2+ stores in downstream IL-1β release, rather than upstream inflammasome activation, may underlie the modest decreases we observed in the IL-1β release responses to ATP, but not nigericin, in the Pam3CSK4- and TNF-α–primed BMDC.

Agonists for some, but not all, Ca2+-mobilizing GPCR expressed in myeloid leukocytes can induce the release of IL-1β via an NLRP3-dependent mechanism(58,59).

Rossol et al. (59) noted that NLRP3 in human and murine monocytes was a target for

CaSR and GPRC6A activated by divalent/trivalent inorganic cations. However, those studies indicated that accumulation of extracellular IL-1β was a delayed response with little cytokine release occurring during the initial 3 h and maximal release requiring >8 h of CaSR activation(59). Lee et al. (58) described more rapid (within 30–50 min) CaSR- induced activation of NLRP3 signaling in murine BMDM and also used the organic

calcimimetic agonist R568 to induce CaSR-dependent NLRP3 inflammasomes. We

compared the relative efficacies of R568 and the K+ efflux agonists to drive rapid NLRP3

inflammasome activation in our LPS-primed BMDC experimental model. Although R568 rapidly induced a sustained increase in cytosolic [Ca2+] during a 30-min test period, this resulted in only very weak accumulation of ASC oligomers that was insufficient to drive significant caspase-1 activation or IL-1β release. Different GPCR have varying rates of desensitization. For example, P2Y2 purinergic receptors desensitize within minutes after activation by the metabolically labile ATP/UTP agonists(122). In contrast, the canonical role of the CaSR is to respond to slowly developing increases in serum extracellular

[Ca2+] for regulation of parathyroid hormone secretion. The receptor is thus remarkably resistant to desensitization and retains the ability to signal via the Gq/PLC cascade with

sustained elevation of cytosolic [Ca2+] for hours after exposure to agonistic stimuli(123).

Sustained elevation of cytosolic [Ca2+] during prolonged CaSR activation may induce

sufficient activity of Ca2+-sensitive K+ channels to decrease cytosolic [K+] to the

threshold level required for stable assembly of NLRP3/ASC signaling platforms. At the

single cell level, the assembly of such stable platforms likely comprises an all-or-none response to coincident combinations of critical regulatory signals, such as decreased cytosolic [K+] as well as the ubiquitination or phosphorylation states of NLRP3 and

ASC(73,74,124). It would be relevant to test whether sustained stimulation of DC or

macrophages with different slowly desensitizing GPCR gradually increases the number

of cells with perinuclear ASC specks and accumulation of active caspase-1.

The Ca2+ chelator BAPTA has been used to implicate increased cytosolic [Ca2+]

as a necessary signal for NLRP3 inflammasome activation in response to various stimuli,

including nigericin and extracellular ATP(56-58,104). Although our studies confirmed the ability of BAPTA loading to markedly attenuate nigericin-induced NLRP3 signaling

in LPS-primed BMDC, this inhibitory action was observed under Ca2+-free stimulation conditions that prevent nigericin-elicited changes in cytosolic [Ca2+]. BAPTA chelates

other trace divalent cations, including Zn2+, Fe2+, and Cu2+, that can function as key cofactors for many enzymes, including some implicated in inflammasome regulation.

Several reports have implicated roles for intracellular Zn2+ in modulation of NLRP3

inflammasome signaling(125). One possible specific link is the zinc-dependent metalloprotease, BRCC3, which acts as a K63-specific deubiquitinase and critical regulator of NLRP3. Py et al. (70) found that BRCC3 promotes NLRP3 inflammasome activation by deubiquitinating the LRR domain of NLRP3. In addition to ubiquitination, other studies have implicated the phosphorylation status of inflammasome components as critical to the efficient assembly of NLRP3/ASC platforms. Martin et al. (73) identified the PP2A phosphatase as a key target for signal 2 stimuli, including ATP and nigericin, which acted to recruit PP2A to complexes of IKKα and ASC. The recruited PP2A reverses association of IKKα with ASC and thus licenses ASC for interaction with

NLRP3. Notably, BAPTA loading attenuated the recruitment of PP2A in response to

ATP or nigericin (73). Previous studies have identified complex roles for cytosolic Zn2+

in regulation of PP2A activity(126,127). Finally, Furuta et al. (106) demonstrated that

BAPTA exerts a potent microtubule-depolymerizing activity that is independent of its ability to chelate Ca2+. This is relevant because Misawa et al. (128) have described a

microtubule-mediated spatial arrangement of mitochondria in close apposition to the ER

that is necessary for optimal NLRP3 inflammasome activation. The ability of BAPTA to

promote microtubule depolymerization might interfere with this organellar juxtaposition

and thus attenuate interaction between ASC and NLRP3. Taken together, the combined

effects of BAPTA on Zn2+-dependent signaling enzymes and/or microtubule dynamics

may underlie its inhibitory actions on NLRP3 signaling independently of its canonical

actions on Ca2+ signaling.

Additional support for the involvement of Ca2+ signaling in NLRP3 activation has

2+ come from experiments using 2-APB as an inhibitor of both ER Ca release via IP3 receptor channels and influx of extracellular Ca2+ via the Orai family Ca2+ release–

activated Ca2+ channels(57,58). We confirmed previously reported observations that

pretreatment of LPS-primed myeloid cells with 2-APB prior to stimulation by nigericin

(or ATP) completely suppresses all indices of the activated NLRP3 signaling cascade.

Importantly, our experiments also demonstrated that 2-APB inhibits the ability of nigericin to induce the pyroptotic propidium2+ influx as an alternative readout of caspase-

1 activation. However, we were unable to correlate these robust inhibitory effects of 2-

APB on NLRP3 signaling with the canonical inhibitory actions of 2-APB on elevation of

cytosolic [Ca2+] and Ca2+ signaling. Rather, 2-APB per se caused increases in cytosolic

[Ca2+] in LPS-primed DC and also facilitated massive influx of Ca2+ during subsequent

stimulation by nigericin (Figure 3.11 E). Although 2-APB does indeed potently block

Ca2+ influx via the Orai1 and Orai2 subtypes of Ca2+ release–activated Ca2+ channels, it

has the opposite effect on the Orai3 family member. Several groups have described the

ability of 2-APB to allosterically stabilize the open-gated conformation of Orai3 channels

and change their permeability properties into nonselective cation channels that facilitate

fluxes of Ca2+ and monovalent cations(129-134). However, Orai3-/- BMDC also

demonstrated 2-APB induced Ca2+ influx, arguing against a role for Orai3 in mediating the increase in cytosolic [Ca2+] observed in response to 2-APB treatment (unpublished

results). Members of the TRPV family of nonselective cation channels (TRPV1, TRPV2

and TRPV3) also open in response to 2-APB(135). Ongoing studies in the lab suggest that the 2-APB sensitive channel in BMDC possesses a pharmacologic profile consistent with TRPV3 (Chapter 5; Figure 5.1). Regardless of how 2-APB induces Ca2+ influx, our

observations indicate that the suppression of NLRP3 inflammasome signaling in 2-APB–

treated BMDC cannot be ascribed to direct inhibition of Ca2+ signaling. 2-APB does not

inhibit the inflammasome signaling cascades initiated by AIM2(58). Given that AIM2, similar to NLRP3, regulates an ASC-dependent inflammasome, this suggests that 2-APB targets NLRP3 function rather than ASC. Additional studies are required to define the underlying pharmacological mechanisms by which 2-APB may inhibit NLRP3 upstream of ASC oligomerization.

In summary, the present study provides new insights regarding how perturbation of intracellular ion homeostasis acts as a signal for activation of NLRP3 inflammasome signaling. The data indicate that a rapid decrease in cytosolic [K+] is a highly efficacious

signal for initiating the NLRP3 inflammasome cascade regardless of the presence or

absence of coincident increases in cytosolic [Ca2+]. Despite this dissociation from Ca2+

signaling, the mechanism by which a decrease in [K+] is coupled to the conformational

activation of NLRP3 remains unknown. Moreover, changes in cytosolic [K+] may also

act downstream of NLRP3 at the level of ASC given the suppressive effects of elevated

[K+] on activation of the AIM2/ASC inflammasome in Francisella-infected

macrophages(136). Perturbation of multiple mitochondrial functions by disruption of the

normal cytosolic [K+]/[Na+] ratio is a relevant area for investigation(114,137). However, a recent analysis by Vince and colleagues (138) demonstrated normal NLRP3

inflammasome function in ATP- or nigerin-stimulated murine macrophages that lacked expression of different mitochondrial or mitochondria-associated proteins, including

cyclophilin D, Bax, Bak, and parkin, previously implicated in the regulation of

inflammasome signaling. These findings indicate that linking NLRP3 activation to

mitochondrial perturbation by K+ efflux agonists will require consideration of other

mitochondrial functions.

Chapter 4: Activation or Inhibition of NLRP3 Inflammasome Signaling by

Lysosome Destabilization is Coordinated by the Extent of Lysosomal

Membrane Permeabilization and Plasma Membrane Cation Channel Activity

Portions of this chapter have been submitted for publication in the Journal of Biological

Chemistry.

Abstract:

NLRP3 is a cytosolic protein that nucleates assembly of inflammasome signaling

platforms which facilitate caspase-1 mediated IL-β release and other inflammatory

responses. NLRP3 inflammasomes are assembled in response to multiple pathogen- or

environmental stress-induced changes in basic cell physiology, including the

destabilization of lysosome integrity and activation of K+-permeable cation channels in

the plasma membrane (PM). However, the quantitative relationships between lysosome

membrane permeabilization (LMP), gating of PM cation channels, and activation of

NLRP3 signaling are incompletely characterized. We used Leu-Leu-O-methyl ester

(LLME), a soluble lysosomotropic agent, to quantitatively track the kinetics and extent of

LMP in relation to the time courses of NLRP3 inflammasome signaling responses (ASC oligomerization, caspase-1 activation, IL-1β release) and PM cation fluxes in murine bone marrow-derived dendritic cells (BMDC). Treatment of BMDC with submillimolar

(<1 mM) LLME induced slowly developing (t½=5-8 min) and partial (<80% max)

increases in LMP that correlated with robust NLRP3 inflammasome activation, K+ efflux,

and Ca2+ influx. In contrast, supramillimolar (>2 mM) LLME elicited extremely rapid

(t½<3min) and complete collapse of lysosome integrity that was coordinated with

blockade of inflammasome signaling and attenuation of PM ion fluxes. Supramillimolar

LLME also induced dominant negative effects on inflammasome activation by the

canonical NLRP3 agonist nigericin. Ca2+ influx attenuated LMP-stimulated NLRP3

inflammasome signaling but potentiated LPM-induced necrosis. Taken together, these studies reveal a previously unappreciated non-linear signaling network that defines the

coupling between LMP, gating of PM cation channels, regulated cell death and NLRP3

inflammasome activation.

Introduction:

IL-1β is a pro-inflammatory cytokine involved in the induction of fever and

mobilization of leukocytes to sites of infection(139). Active IL-1β is produced via

proteolytic processing of pro-IL-1β by caspase-1 (34). Caspase-1 is activated via proximity-induced autocatalysis within macromolecular signaling platforms called inflammasomes (34). A major inflammasome is based on the cytosolic pattern

recognition receptor NLRP3 (34,140). Upon conformational activation, NLRP3 interacts

with the ASC adapter protein to nucleate formation of large cytosolic aggregates known

as ASC specks (37,38). ASC specks form a platform for recruitment of pro-caspase-1

monomers and thereby activate caspase-1 with consequent proteolytic maturation of IL-

1β for release by non-canonical secretion (34,140). Caspase-1 also triggers pyroptosis, a

regulated cell death pathway defined by activation of plasma membrane channels/pores

permeable to osmotically active ions (Na+, K+, Cl-) and small metabolites; this induces

osmotic swelling and and eventual cell lysis (101).

Although the critical biochemical signal(s) that drives conformational activation

of NLRP3 is unknown, NLRP3 inflammasomes are rapidly assembled in response to

multiple pathogen- or environmental stress-induced changes in basic cell physiology,

including ion channel-mediated decreases in cytosolic [K+] and the disruption of

lysosome integrity (35). NLRP3 activation in response to lysosome destabilization

correlates with lysosomal membrane permeabilization (LMP) and release of lysosomal

hydrolases, including multiple cathepsins, into the cytosol (41). LMP is induced by a

broad range of proinflammatory stimuli that include phagocytized particulates, such as

alum, silica, monosodium urate (MSU), and crystalline cholesterol (25,40,112), lipotoxic

saturated fatty acids (15), and soluble lysosomotropic compounds such as the dipeptide

Leu-Leu-O-Me (LLME) (40). Pharmacologic inhibition of cathepsin activity or genomic

deletion of various cathepsins markedly attenuates LMP-induced NLRP3 inflammasome signaling (62,141-143). LMP-mediated activation of inflammasomes markedly contributes to inflammatory disease pathogenesis or progression elicited by some particulate stimuli, including MSU-induced gout (25,144) and crystalline cholesterol- induced atherosclerosis (16,112,145).

The mechanism(s) by which LMP and accumulation of cytosolic cathepsins induces NLRP3 inflammasome complex assembly are incompletely defined. Although direct proteolytic modification of NLRP3 or other inflammasome regulatory proteins may be involved, multiple studies have demonstrated potent suppression of NLRP3 signaling when diverse LMP stimuli are presented to macrophages or dendritic cells (DC) bathed in media containing elevated extracellular [K+] (53,55,62). This prevents the efflux of

cytosolic K+ and consequent decrease in intracellular [K+] that is a necessary (but not

sufficient) signal for NLRP3 inflammasome activation in normal cells which express

non-mutated NLRP3 protein. Moreover, all LMP stimuli tested also trigger robust

decreases in cytosolic K+ prior to, and independently from, NLRP3 inflammasome

assembly and accumulation of active caspase-1 (53). This suggests a model wherein activation of plasma membrane cation channels by lysosome-derived signals (cathepsins,

other hydrolases, or small metabolites) is a critical early signal for licensing NLRP3

inflammasome assembly by LMP stimuli. We recently reported that robust Ca2+ influx

and K+ efflux is rapidly triggered by LLME treatment of murine DC (55).

Although the role of LMP in stimulating NLRP3 inflammasomes is well- established, recent studies by Brojatsch and colleagues have demonstrated that the efficacy of LMP stimuli in activating inflammasome signaling can be very low under some experimental conditions(146-149). Those investigators observed that LLME or particulate alum failed to induce significant caspase-1 processing or IL-1β release in murine macrophages despite causing massive lysosome rupture. The low level of inflammasome signaling in response to LLME and alum correlated with proteolytic degradation of various cytosolic proteins, including pro-caspase-1 and pro-IL-1β, and extensive cytolysis (146). These findings indicate that lysosome destabilization can either activate or suppress NLRP3 inflammasome signaling in different cellular contexts and raise several questions: What quantitative parameters determine the threshold levels of lysosome destabilization to trigger the inflammasome signaling cascade? What percentage of lysosomes must become permeabilized to cause activation of NLRP3?

Might LMP-induced suppression of inflammasome signaling involve mechanisms other than, or in addition to, direct proteolytic degradation of inflammasome proteins?

We addressed these questions using LLME, a soluble lysosomotropic agent, to quantitatively track the kinetics and extent of LMP in relation to the time courses of multiple NLRP3 inflammasome signaling responses and plasma membrane cation fluxes in murine bone marrow-derived dendritic cells (BMDC). We found that submillimolar

LLME induced slowly developing and partial increases in LMP that correlated with robust NLRP3 inflammasome activation, K+ efflux, and Ca2+ influx. In contrast,

supramillimolar LLME elicited extremely rapid and complete collapse of lysosome

integrity accompanied by coordinated suppression or attenuation of inflammasome

signaling and plasma membrane ion fluxes. These results reveal a previously

unappreciated non-linear signaling network biphasic that coordinates the coupling

between LMP, gating of plasma membrane cation channels, entrainment of necrotic

death, and NLRP3 inflammasome activation.

Results:

Leu-Leu-O-methyl ester (LLME) induces concentration-dependent and synchronized

increases in lysosomal membrane permeabilization (LMP) in dendritic cells

As indicated in Chapter 3 (Figure 3.3), treatment of LPS-primed murine BMDC

with 1 mM LLME for 30 min induces robust (~10 ng/ml) release of IL-1β via a

mechanism strictly dependent on NLRP3·ASC inflammasome assembly and increased

K+ efflux (55). The ability of LLME to elicit particularly rapid NLRP3 inflammasome

assembly distinguishes it from the particulate stimuli typically used in studies of LMP-

mediated inflammasome activation. Particulate activators require phagocytosis and

fusion of the particulate-loaded phagosomes with lysosomes. This complex cell biology

will limit the rate and increase the heterogeneity of LMP-dependent responses in DC or macrophage populations, such that lysosome destabilization and NLRP3 activation within the cells will be slower and less synchronized. We reasoned that synchronization of lysosomal destabilization within the cells comprising a BMDC test population would facilitate analysis of the temporal and quantitative relationships between LMP and the

100

extent of NLRP3 activation. LLME is transported into the cytosol via plasma membrane

amino acid transporters and then into the lysosome lumen via lysosomal amino acid

transporters. Lysosomes in leukocytes function as effective sinks for LLME

accumulation due to their particularly high content of cathepsin C, also known as

dipeptidyl peptidase–I (DPP-I). Via its reverse reaction, DDP-I rapidly catalyzes the

conjugation of Leu-Leu dipeptides into Leu4, Leu6, and Leu8 polymers that act as

intraluminal detergents to permeabilize the lysosomal membrane (113).

To verify that LLME can induce rapid, synchronous, and concentration-dependent

lysosome destabilization in our BMDC experimental model, we loaded the cells with 10

kDa FITC-dextran. Dextrans are internalized via endocytosis and trafficked to

lysosomes. In healthy cells, FITC-dextran possesses low fluorescence within the acidic

pH environment of the lysosomal lumen. If only the lysosomal pH gradient is dissipated,

the resulting alkalinization increases fluorescence of FITC-dextran compartmentalized

within lysosomes to produce brighter lysosomal puncta within individual cells and

increased total fluorescence within the cell population. If lysosomal integrity is

completely disrupted, the entrapped FITC-dextran is released into the neutral (pH 7.2) cytosol; this results in bright but non-punctate fluorescence at the single cell level and

increased total fluorescence within the cell population (Figure 4.1 A). We used a plate-

reader to continuously track changes in total fluorescence in FITC-dextan-loaded BMDC monolayers (Figure 4.1 B) and fluorescence microscopy to image FITC-dextran subcellular localization (Figures 4.1 D and 1E). In most LPS-primed but unstimulated

BMDC, FITC-dextran localizes to endosomes/lysosomes as perinuclear punctae with dim fluorescence (Figure 4.1 D, arrow and Figure 4.1 E-1). However, in a minor fraction of

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Figure 4.1: Leu-Leu-O-methyl ester (LLME) induces concentration-dependent and

synchronized increases in lysosomal membrane permeabilization (LMP) in dendritic

cells. A) Schematic of experiments utilizing FITC-dextran to measure lysosome membrane integrity. B) Compromise of lysosome pH gradient was assayed by measuring fluorescence of FITC-dextran loaded BMDC (485ex→528em). Baseline recordings were

taken for 5 min. NG or indicated concentration of LLME was added at t=5 min.

Fluorescence was measured for 30 min subsequent to addition of stimulus. Data are

representative of n=2 independent experiments. C) Slope of curves in B between t=5 min

and t=10 min was calculated for each concentration of LLME. D) Loss of lysosome

membrane integrity was assayed by fluorescence microscopy of FITC-dextran loaded

BMDC. Dotted arrow shows representative cell with partial collapse of lysosome

membrane integrity. Solid triangle shows representative cell with complete loss of

lysosome integrity. E) BMDC were loaded with FITC-dextran and stimulated with NG or various concentrations of LLME for 30 min. Cells were subsequently visualized by fluorescence microscopy. Data are representative of n=2 independent experiments.

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Figure 4.1

103

non-stimulated cells, the FITC-dextran is released into the cytosol to produce a bright but

diffuse pattern of fluorescence which fills the entire cell volume (Figure 1D, arrowhead),

consistent with complete loss of lysosomal membrane integrity. As a positive control, we

observed that nigericin induced a slowly developing (over 15 min) increase in total

fluorescence in the plate reader assay (Figure 4.1 B) and the retention of punctate

labeling but with brighter punctal intensity by cell imaging (Figure 4.1 E-2). This is

consistent with dissipation of the lysosomal transmembrane pH gradient via nigericin’s

K+/H+ exchanger activity, but retention of the 10kD dextran within the lysosomal lumen.

In contrast, stimulation of BMDC with LLME triggered concentration-dependent and

time-dependent increases in total cell fluorescence (Figures 4.1 B and C) that correlated

with increases in cytosolic accumulation of 10 kDa dextan indicative of collapsed

lysosomal integrity (Figures 4.1 E-3 through E-6). Notably, cells treated with 0.5 mM

LLME were characterized by both low level accumulation of cytosolic dextran and retention of bright punctae but at lower numbers. This suggests complete membrane permeabilization in only a subset of lysosomes. The phase contrast images showed large vacuoles consistent with osmotic swelling of lysosomes. 1 mM LLME induced a more rapid, but still submaximal, increase in total fluorescence (Figure 4.1 B) and higher cytosolic dextran levels but with some retention of punctate labeling. Stimulation of

BMDC with 2 or 3 mM LLME for 30 min resulted in complete collapse of most lysosomes, as evidenced by bright cytosolic staining (Figures 4.1 E-5 and E-6) in the majority of cells and very rapid (t ½ <2 min) increases in total fluorescence (Figures 4.1 B

and C).

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NLRP3 inflammasome signaling is activated by low-level LMP but inhibited by high-level

LMP

The Figure 4.1 data indicate that LLME elicits rapid and synchronous increases in

LMP which are graded in magnitude depending on [LLME]. We used identical

experimental conditions (e.g., 30 min test periods) and the same range of LLME

concentrations to characterize LMP-triggered NLRP3 inflammasome signaling, as indicated by IL-1β release, in LPS-primed BMDC (Figure 4.2 A). A bell-shaped

concentration-response relationship defined the effects of LLME on IL-1β release with a

peak response at 1 mM, followed by decreasing cytokine release with increasing LLME.

The ability of submillimolar LLME to stimulate IL-1β release reflected inflammasome

signaling and was absent in caspase-1-deficient BMDC. Notably, the maximal IL-1β release (8-10 ng/ml) observed with 1 mM LLME was invariably 2-fold lower in magnitude than that (16-30 ng/ml) triggered by nigericin in parallel samples of the same

BMDC preparations (Figure 4.2 A). These findings indicate that the slower, partial LMP

(Figure 4.1) induced by submillimolar LLME generates stimulatory signals for NLRP3 activation, but that these signals are antagonized or reversed by the more robust LMP triggered by supramillimolar LMP. Kinetic analysis verified that the attenuated IL-1β release observed with supramillimolar LLME was not due to a slower rate of inflammasome activation; for all [LLME]>1 mM, no additional IL-1β release was

observed after 30 min (Figure 4.2 B).

Disruption of lysosome membrane integrity can trigger a caspase-independent

mode of necrotic cell death. This lysosome-dependent pathway, termed LMP-induced cell death, is characterized by disruption of plasma membrane integrity (94). In

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Figure 4.2: NLRP3 inflammasome signaling is activated by low-level LMP but inhibited by high-level LMP. A) IL-1β release from LPS-primed WT or caspase-1-/-

BMDC was measured by ELISA in response to stimulation with various concentrations of LLME at 30 min time point. Data represent a mean of n=4 independent experiments.

B-D) LPS-primed WT BMDC were stimulated with 10µM NG or various concentrations of LLME for 2 hr time course. B) IL-1β release was measured by ELISA. Data represent a mean of n=2 independent experiments and are representative of n=4 independent experiments. C) Necrotic cell death was quantified by measurement of LDH activity in extracellular medium. Data represent a mean of n=4 independent experiments. D) LDH release in response to various concentrations of LLME at 30 min time point from experiment in panel C. E-F) LPS-primed WT BMDC were stimulated with various combinations of NG (10µM) and LLME (1mM or 5mM). E) IL-1β release was assayed by ELISA. Data represent a mean of n=2 independent experiments. F) Formation of ASC filaments, caspase-1 processing, and IL-1β processing/release were assayed by western blot.

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Figure 4.2

107

inflammasome-competent cells, such as BMDC, LMP-induced cell death will additionally reflect caspase-1-dependent pyroptosis. LLME induced time-dependent

(Figure 4.2 C) and concentration-dependent (Figure 4.2 D) increases in release of the cytosolic enzyme lactate dehydrogenase (LDH) as an indicator of lytic cell death.

Maximal LDH release at each [LLME] (or with nigericin) required >60 min of stimulation (Figure 4.2 C). In contrast to the bell-shaped concentration/response curve for IL-1β release (Figure 4.2 A), LLME-induced LDH release was characterized by conventional hyperbolic dependence on [LLME] with a plateau at > 1mM (Figure 4.2 D).

We tested whether the reduced efficacy of supramillimolar LLME in stimulating

IL-1β release reflected deficient assembly of the NLRP3 inflammasome complex. If the

robust LMP triggered by high [LLME] alters the expression levels or function of one or

more of the members of this complex (NLRP3, ASC, caspase-1, or pro-IL-1β), this may

suppress inflammasome activation by other NLRP3 agonists such as nigericin. Indeed,

co-treatment of BMDC with 5 mM LLME prevented the robust IL-1β release response to nigericin (Figure 4.2 E). Consistent with the bell-shaped [LLME]-IL-1β release response curve, 5 mM LLME by itself elicited minimal IL-1β secretion. Interestingly, cells co- treated with 1 mM LLME plus nigericin released the same amount of IL-1β as cells stimulated with only 1 mM LLME, rather than the 2-fold greater amount of cytokine elicited by nigericin alone (Figure 4.2 E). We also observed suppressed processing/release of caspase-1 and IL-1β by western blot analysis of lysates and conditioned medium from BMDC co-treated with 5 mM LLME versus nigericin alone

(Figure 4.2 F). The western blots confirmed that 1 mM LLME alone triggers caspase-1 activation and IL-1β processing but prevents the even more robust responses induced by

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nigericin alone. LLME alone, at 1 but not 5 mM, induced strong ASC oligomerization

albeit less intense than observed in BMDC stimulated with nigericin (Figure 4.2 F). As

with the other indices of NLRP3 inflammasome signaling, 5 mM LLME completely

prevented nigericin-induced ASC oligomerization in co-treated cells, while co-treatment

with 1 mM LLME plus nigericin yielded ASC oligomerization intensity similar to that

produced by 1 mM LLME alone.

Lima et al. (146) reported that weak NLRP3 inflammasome signaling responses

in murine macrophages treated for 2 h with 2.5 mM LLME correlated with cathepsin-

mediated degradation of procaspase-1 and proIL-1β. In contrast, treatment of BMDC with 5 mM LLME for 30 min in our experimental model did not result in obvious decreases in intracellular levels of procaspase-1, proIL-1β, monomeric ASC, or actin despite the prevention of ASC oligomerization, procaspase-1 autocatalytic processing,

IL-1β maturation, and the release of mature IL-1β and the p20 subunit of active caspase-1

(Figure 4.2 F). Taken together, the Figure 4.2 experiments indicate that NLRP3 inflammasome assembly is activated by lysosome-derived factors released or produced

during submaximal or “low-level” LMP. However, these stimulatory signals are

antagonized by other lyosomal signals or factors which rapidly accumulate during the

massive lysosomal disruption (“high-level LMP”) induced by supramillimolar LLME.

Plasma membrane cation channels and pyroptotic pores are activated by low-level LMP

but attenuated by high-level LMP

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We (55) and others (53) have established a critical role for decreased cytosolic

[K+] in the activation of NLRP3 inflammasomes by LMP stimuli. Although LMP-

induced K+ efflux may be mediated by as yet unidentified selective K+ channels, our

results in Figure 3.3 D indicated that stimulation of BMDC with 1 mM LLME also

caused a rapid influx of extracellular Ca2+(55). The ability of LLME to stimulate both

K+ efflux and Ca2+ influx suggests that LMP may trigger parallel gating of Ca2+ channels,

K+ channels, and/or non-selective cation channels in the plasma membrane. We assayed

the rate of increase in cytosolic [Ca2+] as an indicator of potential non-selective cation channel activity in both wildtype (Figure 4.3 A) and caspase-1 knockout (Figure 4.3 B)

BMDC stimulated with same range of LLME concentrations used in the assays of LMP

(Figure 4.1) and inflammasome signaling (Figure 4.2). After a ~3 min lag, all

concentrations of LLME triggered increases in cytosolic [Ca2+]. Remarkably, LLME- induced Ca2+ influx rates in both wildtype and caspase-1 deficient BMDC were

characterized by bell-shaped concentration-response relationships (Figure 4.3 C) similar

to that characterizing LLME-induced IL-1β release (Figure 4.2 B). As for IL-1β release,

1 mM LLME stimulated the greatest response. This was characterized by increasing Ca2+

influx over 10 min to reach a steady-state cytosolic [Ca2+] of ~750 nM that was 10-fold

higher than the basal level. Removal of extracellular Ca2+ greatly reduced the LLME-

elicited increases in cytosolic [Ca2+] indicating a predominant role for Ca2+ influx rather

than mobilization of intracellular Ca2+ stores (Figure 3.3 D and (55)). By comparison, the Ca2+ ionophore ionomycin elicited near-immediate ~50-fold increases in cytosolic

[Ca2+] in both wildtype and caspase-1 deficient cells. These findings suggest that LLME-

induced LMP triggers the gating of Ca2+-permeable cation channels in the plasma

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Figure 4.3: Plasma membrane cation channels and pyroptotic pores are activated by

low-level LMP but attenuated by high-level LMP. A and B) Changes in cytosolic

[Ca2+] were analyzed by measurement of fluo-4 fluorescence. WT or caspase-1-/- BMDC

were loaded with 1µg/ml fluo-4 AM for 30 min. Baseline readings were taken for 5 min.

Cells were stimulated with 6µM ionomycin or various concentrations of LLME at t=5

min. Data represent a mean of n=4 independent experiments for WT BMDC and n=4

independent experiments for caspase-1-/- BMDC. C) Slope of curves from panels A and

B between t=10 min and t=20 min. D and E) LPS-primed WT or caspase-1-/- BMDC

were stimulated with 10µM NG or various concentrations of LLME for 30 min. Onset of

cell death in response to NG or LLME was quantified by fluorescence measurement of

propidium2+ influx. Data represent a mean of n=2 independent experiments for WT

BMDC and n=2 independent experiments for caspase-1-/- BMDC. F) Slope of curves

from panels D and E between t=25 min and t=15 min. G) Induction of pyroptotic cell

death in response to NG was assayed by fluorescence measurement of propidium2+ influx.

Baseline readings were taken for 5 min. LLME was added to relevant wells at t=5 min

and NG was added to relevant wells at t=15 min. Data represent a mean of n=2

independent experiments. H-I) LPS-primed caspase-1-/- BMDC were treated with various

concentrations of LLME for 30 min (H) or LPS-primed WT BMDC were treated with

various concentrations of LLME for 10 min (I). Cells were lysed with 10% nitric acid

and potassium content of lysates was analyzed using atomic absorption spectroscopy.

Data represent a mean of n=4 independent experiments for panel H and n=3 independent

experiments for panel I.

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Figure 4.3

112

membrane as a very early signaling response upstream of caspase-1 activation. However,

as for inflammasome activation, the putative channels are optimally activated by signals

generated during submaximal LMP, but antagonized or blocked by inhibitory signals

produced during rapid and complete disruption of the lysosome pools within BMDC.

Activation of NLRP3 and other inflammasome platforms also triggers pyroptotic cell death. A critical signal for initiation of the pyroptotic cascade is the caspase-1- mediated induction of plasma membrane channels or pores permeable to the major inorganic osmolytes (e.g., Na+, Cl-, K+) and small osmotically active metabolites.

Opening of these pyroptotic channels leads to osmotic cell swelling which, if sustained,

results in cytolysis. We and others have shown that pyroptotic channels are also

permeable to cationic DNA-intercalating dyes, such as propidium2+ (Mr 416 Da), which

can used to kinetically track the caspase-1-dependent opening of the channels in dendritic cells or macrophages under experimental conditions identical to those in the assays of

lysosome disruption (Figure 4.1 B) inflammasome signaling (Figures 4.2 A, B, E and F),

or Ca2+ influx (Figures 4.3 A and B). Consistent with our observations in Chapter 3

(Figure 3.1), nigericin-stimulated propidium2+ influx was initiated after a 10-12 min lag phase in wildtype BMDC (Figure 4.3 D) and was absent in caspase-1-/- BMDC (Figure

4.3 E). Treatment of BMDC with increasing concentrations of LLME also induced

propidium2+ influx in both wildtype caspase-1 deficient BMDC after a 10 min lag time.

However, LLME-induced-propidium2+ influx was defined by a bell-shaped

concentration-response relationship in wildtype cells but a near-hyperbolic curve in the

cells lacking caspase-1 (Figure 4.3 F). These results indicate that LLME-induced LMP

can trigger propidium2+ uptake both via caspase-1-dependent pyroptotic channels and a

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caspase-1 independent mechanism. Notably, the hyperbolic concentration-response curve

characterizing LLME effects on propidium2+ uptake in caspase-1 knockout cells (Figure

4.3 F) that was similar to the [LLME]-LDH release concentration-response curve (Figure

4.2 D).

Increased propidium2+ fluorescence can reflect its influx via activated plasma

membrane channels followed by intercalation with nuclear DNA (or double-stranded

regions of cytosolic RNA), but also direct binding to extracellular DNA that accumulates

secondary to cytolysis. As noted previously, collapse of lysosome membrane integrity

triggers a caspase-independent form of necrotic cell death (94) involving permeabilization of the plasma membrane. Regardless of mechanism, the Figure 4.3 E data indicate that all tested concentrations of LLME induced a slowly developing increase in propidium2+/DNA interaction in the caspase-1-deficient BMDC. In wildtype cells, this response will be additive with propidium2+ uptake via caspase-1-dependent

pyroptotic channels induced by [LLME] < 1 mM that license lower-level LPM.

Consistent with the ability of supramillmolar LLME to suppress nigericin-induced

inflammasosome activation (Figures 4.2 E and F), we found that pretreatment BMDC with 5 mM, but not 1 mM, LLME inhibited nigericin-activation of pyroptotic channel opening (Figure 4.3 G).

LLME treatment also triggered rapid and concentration-dependent decreases in

cytosolic [K+] in both wildtype (Figure 4.3 I) and caspase-1-deficient (Figure 4.3 H)

BMDC. The maximal decrease in [K+] was induced by 1 mM LLME but the magnitude

was less than that elicited by nigericin. Thus, the lower efficacy of LLME relative to

nigericin as an activator of IL-1β release (Figure 4.2 A) correlates with its lower efficacy

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as an activator of K+ efflux. However, in contrast to Ca2+ influx, LLME-stimulated K+

efflux was not characterized by a biphasic concentration-response relationship; this

suggests that different channels may mediate Ca2+ influx and K+ efflux.

Influx of extracellular Ca2+ during lysosome destabilization attenuates LLME-induced

NLRP3 inflammasome signaling

As indicated in the studies described in Chapter 3, increased cytosolic [Ca2+] is

neither a sufficient nor necessary signal for inflammasome activation by the canonical

NLRP3 agonists, nigericin and extracellular ATP. We additionally observed that removal

of extracellular Ca2+ enhanced IL-1β release in response to 1 mM LLME (Figure 3.3 C)

and (55). This suggested that influx of extracellular Ca2+ may have a selective inhibitory

or restraining action on NLRP3 inflammasome activation by LMP stimuli. Experiments

in Figure 4.4 tested this by examining multiple readouts of inflammasome signaling in

LPS-primed BMDC treated with increasing [LLME] in Ca2+-free media versus media with normal (1.5 mM) extracellular Ca2+. Ca2+-free conditions also enhanced the magnitude of IL-1β release in response to all tested [LLME] (Figures 4.4 A and E); thus,

the bell-shaped concentration-response relationship observed in Ca2+-containing saline

was retained but with enhanced efficacy of LLME. The ELISA results were confirmed by

western blot analysis showing: 1) enhanced production and release of active caspase-1

p20 subunit and mature IL-1β (Figure 4.4 D); and 2) greater accumulation of ASC

oligomers (Figure 4.4 E) over the entire range of [LLME] when tested in the absence of

extracellular Ca2+. The presence or absence of Ca2+ did not markedly alter intracellular

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Figure 4.4: Influx of extracellular Ca2+ during lysosome destabilization attenuates

LLME-induced NLRP3 inflammasome signaling. A-F) LPS-primed WT BMDC were stimulated with various concentrations of LLME for 30 min in the presence/absence of extracellular Ca2+. A) IL-1β release was quantified by ELISA. Data represent a mean of

n=5 independent experiments. B) Compromise of lysosome pH gradient in the absence of

extracellular Ca2+ was assayed by measuring fluorescence of FITC-dextran loaded

BMDC (485ex→528em). After loading with FITC-dextran, cells were transferred into

Ca2+-free extracellular medium and baseline recordings were taken for 5 min. NG or

indicated concentration of LLME was added at t=5 min. Fluorescence was measured for

30 min subsequent to addition of stimulus. Data are representative of n=2 independent

experiments. C) Slope of FITC-dextran fluorescence curves from panels 1B

([Ca2+]=1.5mM) and 4B ([Ca2+]=0mM) were calculated between t=10 min and t=5 min.

D) Caspase-1 processing and IL-1β processing/release were assayed by western blot. E)

ASC filament formation was assayed by western blot of DSS-crosslinked insoluble cell

lysate fraction. F) NLRP3 level in cell lysate was assayed by western blot. Panels D-F

are representative of n=2 independent experiments.

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Figure 4.4

117

expression levels of procaspase-1 (Figure 4.4 D), proIL-1β (Figures 4.4 D and E), ASC

(Figure 4.4 D) or NLRP3 protein (Figure 4.4 E) during these 30 min treatments with

LLME. (In one of three similar experiments, 5 mM LLME did decrease NLRP3 protein

by >50%, data not shown). Importantly, removal of extracellular Ca2+ did not attenuate,

but modestly accelerated, the LLME-induced LMP responses observed in FITC-dextran

BMDC (Figure 4.4 B and 4.4 C). Taken together, the experiments in Figure 4.4 indicate

that the influx of extracellular Ca2+ which accompanies LLME-induced LMP attenuates

the assembly of NLRP3 inflammasomes and consequent proteolytic maturation of

caspase-1 and IL-1β.

Influx of extracellular Ca2+ during lysosome destabilization potentiates LLME-induced

cell death

The experiments in Figures 4.3 D-F indicate that LLME-induced LMP correlates with activation of propidium2+ uptake via both caspase-1-dependent and independent

mechanisms. This propidium2+ accumulation occurs prior to, or coincident with, the onset

of LLME-induced cytolysis indicated by LDH release (Figures 4.2 C and D).

Propidium2+ influx in wildtype cells reflects in part the caspase-1 activated

channels/pores that initiate the pyroptotic death cascade. In contrast, the signals that

mediate the caspase-1-independent pathway of LMP-induced cell death are poorly

characterized (94). Given 1) the known roles for Ca2+ influx in other modes of non-

apoptotic cell death (150-153); and 2) the modulating action of Ca2+ influx on LMP-

activated NLRP3 inflammasome signaling (Figure 4.4), we tested how Ca2+ influx might

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contribute to LMP-induced death signaling in BMDC. Because Figure 4.2 C indicated

that LLME-induced LDH release was near-maximal after 60 min, we compared the concentration-response relationships in wildtype and caspase-1 deficient (Figure 4.5 B)

BMDC treated for 1 h with increasing [LLME] in the presence or absence of extracellular

Ca2+ (Figure 4.5 A). Figure 3.3 D demonstrated that LLME-induced increases in

cytosolic [Ca2+] are completely suppressed in the absence of the normal 1.5 mM

extracellular CaCl2 (55). There were no significant differences in the LLME-induced

LDH release responses between wildtype and caspase-1 deficient cells when stimulated

in standard Ca2+-containing medium. In contrast, LLME-elicited LDH release from both

DC genotypes was markedly attenuated in the absence of extracellular Ca2+. The cytoprotective effect of the low Ca2+ stimulus condition was particularly pronounced in

caspase-1 knockout cells as indicated by the >5-fold decreases in the magnitude of LDH release.

The absence of extracellular Ca2+ also attenuated the rate of propidium2+

accumulation in both cell genotypes treated with 1 or 3 mM LLME (Figures 4.5 B-D).

As with the LDH release response, the ability of low Ca2+ to sustain a normal plasma

membrane permeability barrier to propidium2+ was most efficacious in caspase-1- deficient BMDC during LMP triggered by 1 mM LLME (Figure 4.5 B). This is consistent with the Figure 4.3 F data which indicated that ~30% of the propidium2+ influx induced

by 1 mM LLME is mediated by caspase-1-activated pyroptotic pores. The combination

of caspase-1 deficiency and absence of extracellular Ca2+ synergistically reduced the rate

of propidium2+ uptake elicited by 1 mM LLME. Because 3 mM LLME attenuates

NLRP3 inflammasome assembly, propidium2+ accumulation under these test conditions

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Figure 4.5: Influx of extracellular Ca2+ during lysosome destabilization potentiates

LLME-induced cell death. A and B) LPS-primed WT or caspase-1-/- BMDC were

stimulated with various concentrations of LLME for 30 min in the presence/absence of

extracellular Ca2+. Necrotic cell death was quantified by measurement of LDH activity in

extracellular medium. Data represent a mean of n=4 independent experiments for WT

BMDC and n=4 independent experiments for caspase-1-/- BMDC. C and D) LPS-primed

WT or caspase-1-/- BMDC were stimulated with 10 µM NG or various concentrations of

LLME for 30 min in the presence/absence of extracellular Ca2+. Changes in plasma

membrane permeability in response to LLME were quantified by fluorescence

measurement of propidium2+ influx. Data represent a mean of n=2 independent

experiments for WT BMDC and n=2 independent experiments for caspase-1-/- BMDC.

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Figure 4.5

121

is predominated by the Ca2+-sensitive non-pyroptotic pathway, and is thus equivalent in

wildtype and caspase-1-deficient BMDC. Consistent with our findings in Chapter 3 (55),

the absence of extracellular Ca2+ increased pyroptotic pore-mediated propidium2+ influx in response to nigericin (Figures 4.5 B and C). In contrast to LLME, nigericin induces no caspase-1 independent propidium2+ influx (even after 2 h as we have recently reported

(154)), in the presence or absence of extracellular Ca2+ (Figures 4.5 B and D). These

results indicate that influx of extracellular Ca2+ is a critical signaling event in the

initiation of LMP-induced cell death independent of caspase-1-mediated pyroptosis.

TRPM2 cation channels are not major regulators of LMP-induced NLRP3 inflammasome

signaling in BMDC but contribute to LMP-activated K+ efflux

TRPM2 channels are expressed in many cell types including myeloid leukocytes

(155). Zhong et al. reported that LMP stimuli (e.g., phagocytosis of alum and silica

crystals) induce mitochondrial ROS production in macrophages with consequent gating

of ROS-sensitive TRPM2 nonselective cation channels measured by Ca2+ influx (62).

Inhibition or knockout of TRPM2 channels caused a ~50% reduction in NLRP3 inflammasome signaling. Although K+ efflux was not measured in that study, TRPM2 is

known to function as a non-selective cation channel with significant K+ permeability

(61,156). Thus, we tested whether TRMP2 contributes to LLME-induced K+ efflux and

NLRP3 inflammasome signaling by comparing the responses in wildtype and TRPM2

deficient BMDC. The latter DC were isolated from a TRPM2-deficient mouse strain that has been extensively characterized in various in vivo models of metabolic stress-induced

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tissue dysfunction, including cardiac and renal ischemia-reperfusion injury (157,158).

Figure 4.6 A shows that TRPM2 deficiency attenuated the magnitude of the 1 mM

LLME-induced [K+] decrease by ~25% in the either presence or absence of the normal

+ 1.5 extracellular CaCl2. By comparison, nigericin-stimulated K efflux was similar in

+ both BMDC genotypes. We also measured K efflux in cells treated with 1 mM H2O2, a

commonly used extrinsic redox stress stimulus for TRPM2 channel activation.

Experiments indicated that ~200 µM H2O2 was the threshold concentration for inducing

2+ 2+ Ca influx and 1 mM H2O2 produced the maximal rate of Ca influx in BMDC (data not

+ shown). Figure 4.6 A illustrates that H2O2 induced a decrease in cytosolic [K ] in the

wildtype DC but with much lower efficacy compared to nigericin or LLME; this response

to H2O2 was attenuated by ~40% in the TRPM2-deficient cells. Comparative

2+ measurements of Ca influx activated by 1 mM LLME or 1 mM H2O2 in both wildtype

(Figure 4.6 B) and TRPM2-deficient (Figure 4.6 C) cells also indicated that TRPM2

mediates only a part of this response. Taken together, these direct assays of K+ efflux and Ca2+ influx indicate that plasma membrane channels other than TRPM2 are the major

targets for LMP-dependent perturbation of cation homeostasis in BMDC. Accordingly,

the TRPM2-deficient dendritic cells were characterized by only modest decreases

(lacking statistical significance) in LLME-induced processing and release of IL-1β as

measured by ELISA (Figure 4.6 D) or western blot (Figure 4.6 E). TRPM2 deficient

BMDC exhibited robust nigericin-activated caspase-1 cleavage (Figure 4.6 E), IL1-β

processing (Figures 4.6 D and E) and induction of pyroptotic channels (Figure 4.6 F).

Interestingly, H2O2 treatment failed to stimulate any inflammasome signaling responses

in either wildtype or TRPM2-deficient BMDC (Figures 4.6 D and E). Thus, TRPM2

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Figure 4.6: TRPM2 cation channels are not major regulators of LMP-induced

NLRP3 inflammasome signaling in BMDC but contribute to LMP-activated K+

efflux. A and D) LPS-primed WT or TRPM2-/- BMDC were stimulated with 10 µM NG

for 30 min, 1mM LLME for 30 min, or 1 mM hydrogen peroxide (H2O2) for 3 hrs.

Where indicated, BMDC in Ca2+-free saline were also stimulated with 1 mM LLME. A)

Cells were lysed with 10% nitric acid and K+ in lysates was analyzed using atomic

absorption spectroscopy. Data represent a mean of n=2 independent experiments. B and

C) Changes in cytosolic [Ca2+] were analyzed by fluo-4 fluorescence. LPS-primed WT or

TRPM2-/- BMDC were loaded with 1µg/ml fluo-4 AM for 30 min. Baseline readings

were taken for 5 min. Cells were stimulated with 1 mM LLME or 1 mM H2O2 at t=5 min.

Recordings are from a single experiment representative of two independent experiments.

D) IL-1β release was assayed by ELISA. Data represent a mean of n=2 independent

experiments. E) LPS-primed WT or TRPM2-/- BMDC were stimulated with 10 µM NG, 1 mM LLME, or 1 mM H2O2 for 30 min. Intracellular procaspase-1 and proIL-1β and extracellular p20 caspase-1 subunit and mature IL-1β were assayed by western blot. F)

Activation of pyroptotic pores in response to 10µM NG was assayed propidium2+ influx

in LPS-primed WT or TRPM2-/- BMDC. NG was added at t=0 min and fluorescence was

recorded for 45 min. Data represent the average of duplicates from a single experiment.

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Figure 4.6

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likely acts in conjunction with other non-selective cation channels to mediate the K+

efflux required for LMP-induced inflammasome signaling.

Discussion

This study provides several new insights regarding the proximal signaling

mechanisms that couple lysosome membrane permeabilization (LMP) to the assembly of

NLRP3 inflammasomes in myeloid leukocytes. First, the study extends previous findings

which indicate that changes in plasma membrane cation fluxes comprise one of the

earliest (within minutes) responses to LMP. These cation fluxes include both K+ efflux, which is critically permissive for NLRP3 activation, and Ca2+ influx, which attenuates

NLRP3 activation in the context of LMP. Second, we identified a biphasic relationship between the extent of LMP and the efficacy of NLRP3 activation such that inflammasome signaling is stimulated by low-level LMP, but rapidly and efficiently inhibited by massive lysosome disruption. Co-stimulation experiments indicated that the inhibitory signals generated by maximal LMP act in a dominant-negative manner to prevent ASC oligomerization by the canonical NLRP3 agonist nigericin. Third, the

LMP-induced trans-inhibition of nigericin-stimulated NLRP3 inflammasome signaling occurred in the absence of, or prior to, any major decreases in the protein levels of

NLRP3, ASC, procaspase-1, or proIL-1β. This suggests that the LMP-generated inhibitory signals may target acute covalent modifications (e.g., ubiquitination or phosphorylation) in NLRP3 or ASC required for efficient NLRP3•ASC complex formation. Fourth, the study extends recent studies indicating that lysosome disruption

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can trigger either caspase-1-dependent or caspase-1-independent regulated cell death by showing that LMP-induced Ca2+ influx is a critical signal for the caspase-1-independent death pathway in myeloid leukocytes. Taken together, these new observations suggest a model (Figure 4.7) whereby a non-linear signaling network defines the coupling between

LMP, gating of PM cation channels, regulated cell death and NLRP3 inflammasome activation.

LMP-generated signals for the activation of plasma membrane ion channels/transporters

Decreased cytosolic [K+] is an obligatory signal for activation of NLRP3 inflammasome signaling by all tested LMP stimuli, including crystalline particulates and

LLME (53,55). However, the mechanism(s) by which perturbation of lysosome membrane integrity is coupled to changes in plasma membrane function are largely undefined. Observations from this study and Chapter 3 (55) indicate that LLME-induced

LMP causes maximal increases in both Ca2+ influx and K+ efflux within 5-10 min

independently of NLRP3 and caspase-1 activation. However, K+ efflux was defined by a

simple hyperbolic dependence on [LLME] (Figures 4.3 H and I) while a bell-shaped

dose-response characterized LLME-activated Ca2+ influx (Figure 4.3 C). These distinct

concentration-response relationships strongly suggest that Ca2+ influx and K+ efflux are

mediated in part by different and/or differentially regulated plasma membrane ion

channels. The marked suppression of Ca2+ influx with increased LMP also argues against

non-selective plasma membrane permeabilization or cell lysis as an underlying

mechanism at least at early (< 15 min) time points following massive LMP. An

important area for future studies is to define the molecular identities of plasma membrane

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Figure 4.7: Model of biphasic concentration dependence of NLRP3 activation in response to lysosome destabilization.

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ion channels/ transporters activated by LMP. Several TRP family cation channels are

highly expressed in myeloid leukocytes; these include TRPM2, TRPV2, and TRPM7

which are characterized both by differential Na+:K+:Ca2+ permeability ratios and

differential gating by various metabolic or cellular stress signals. We found that TRPM2

channels mediate only a modest fraction of the LLME-induced K+ efflux and Ca2+ influx

responses in BMDC (Figures 4.6 A-C). In contrast to a previous study in bone marrow

macrophages, TRPM2 channels in dendritic cells did not contribute significantly to LMP-

induced NLRP3 inflammasome activation. This indicates that multiple species of plasma

membrane ion channels (TRP-family or non-TRP family) which are variably expressed in

different subsets of myeloid leukocytes may be targeted by lysosome-derived signals as

part of the LMPNLRP3 signaling pathway. Our complementary kinetic

characterization of LLME-induced lysosome disruption and Ca2+ influx indicated only a

brief lag time (<5 min) between cytosolic accumulation of lysosomal macromolecules

and increased plasma membrane ion fluxes. This may be consistent with direct gating of

plasma membrane-resident cation channels by lysosomal proteases or lysosome-derived

second messengers. This temporal relationship is also consistent with potential trafficking

of ions channels from intracellular organelles to the plasma membrane. In this regard, a

recent study indicated that lysosome-resident TRPML3 cation channels sense

neutralization of lysosomal pH (by internalized bacteria) to stimulate exocytotic fusion of

the neutralized lysosomes with the plasma membrane (159). Thus, future studies should test whether LMP-induced changes in plasma membrane cation flux also involve increased trafficking of the normally lysosome-resident mucolipin TRP-family channels that include TRPML1, 2 and 3 (156).

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LMP-generated signals for the activation of NLRP3 inflammasome signaling

Disruption of lysosome integrity induced by internalized crystalline particles,

insoluble protein aggregates, or lysosomotropic small molecules is recognized as a major

cellular stress stimulus for initiation of NLRP3 inflammasome-mediated proinflammatory response (41,160). However, the reported efficacy of different lysosome destabilizing agents as inflammasome stimuli varies widely. Hornung et al. (40) first characterized

LLME as an NLRP3 activator by demonstrating that 0.5-1mM LLME elicited release of ng/ml levels of IL-1β in BMDM. In contrast, Brojatsch and colleagues reported only minor IL-1β production in BMDM treated with LLME in the 2.5-20 mM range(146). Our kinetic analyses in a BMDC model resolve these disparate observations by demonstrating that bell-shaped concentration-response relationships describe the effects of LLME on multiple readouts of NLRP3 inflammasome signaling including ASC oligomerization, caspase-1 autocatalytic processing and release, IL-1β maturation and release, and induction of pyroptotic channels/pores.

Although the signaling pathway linking lysosome permeabilization to activation of NLRP3 remains an area of active investigation, multiple studies have indicated that the proteolytic activity of lysosomal cathepsins is required for inflammasome assembly in response to particulates and LLME (40,112). Cathepsins are released into the cytosol by lysosome destabilizing agents and pharmacologic cathepsin inhibitors such as CA-074 prevent inflammasome complex assembly downstream of LMP. Orlowski et al. have recently reported that the proteolytic activity of multiple redundant cathepsins is required for NLRP3 inflammasome activation in response to LMP-inducing particulates or LLME

(143). Specifically, the enzymatic activities of cathepsins B, L, C, S and X are involved

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in mediating LMP-induced inflammasome assembly. Genomic knockdown of one

cathepsin does not inhibit IL-1β release in response to particulates/LLME because cells

respond by upregulating expression of the remaining cathepsins. A significant decrease in

LMP-induced IL-1β release was observed only with a compound genomic knockdown of

all five cathepsin proteases described above or with use of pharmacologic compounds

that inhibit the activity of multiple cathepsins. Interestingly, that study also

demonstrated that cathepsin activity is required for NF-κB mediated synthesis of pro-IL-

1β (143). Incubation of macrophages with pharmacologic cathepsin inhibitors decreased

synthesis of pro-IL-1β in response to TLR4 activation.

LMP-generated signals for the suppression of NLRP3 inflammasome signaling

Our findings suggest that an inhibitory factor(s) is released or produced upon

extensive lysosomal disruption to attenuate NLRP3 inflammasome activation or

assembly (Figure 4.7). This factor does not cause degradation of inflammasome

components (NLRP3, ASC, pro-caspase-1, pro-IL-1β) (Figure 4.4 D and E). Rather, it appears to exert a “dominant negative” inhibitory effect on the inflammasome machinery and prevents inflammasome assembly in response to the K+ efflux agonist nigericin

(Figure 4.2 E and F). Inflammasome components are regulated at the post-translational level and the lysosomal inhibitory factor may interfere with one or several of these post-

translational modifications. One study reported that NLRP3 is ubiquitinated in the basal

state and must be deubiquitinated by BRCC3 to facilitate regulation by “signal 2”

activators (70). Another report indicated that ASC must be linearly ubiquitinated by

LUBAC to mediate efficacious inflammasome signaling (76). The lysosomal inhibitory

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factor may interfere with these or other ubiquitin modifying enzyme complexes. ASC is

also regulated by the activity of kinases and phosphatases. In LPS-primed myeloid cells,

ASC is localized to the perinuclear space and associates with IKKα (73).

Dephosphorylation of ASC by PP2A and dissociation from IKKα has been linked to active inflammasome complex assembly (ASC specks) (73). The putative inhibitory factor released from compromised lysosomes may modulate ASC dissociation from

IKKα and/or dephosphorylation by PP2A.

LLME also induced robust Ca2+ influx within 5 min (Figure 4.3 A and B). Recent reports have indicated that increased cytosolic [Ca2+] either activates or has no

significant modulatory effect on NLRP3 inflammasome activation in different

experimental models (55,57). In contrast, results illustrated in Figure 4.4 demonstrate that

increased cytosolic [Ca2+] inhibits ASC filament formation triggered by high-level LMP.

This inhibitory action of elevated [Ca2+] at a very proximal step in the inflammasome

signaling cascade suggests effects at: 1) conformation activation of NLRP3 per se; 2) the

association of NLRP3 with ASC; or 3) the process of ASC filament assembly.

Lima et al. found that treatment of BMDM with 2.5mM LLME for 2 h resulted in proteolytic degradation of many cytosolic proteins, including pro-IL-1β and pro-caspase-

1 (146). Although we observed that a 30 min incubation of BMDC with [LLME]>1mM strongly suppressed NLRP3 inflammasome assembly and signaling, it did not decrease levels of the key inflammasome proteins (NLRP3, ASC, pro-caspase-1, pro-IL-1β)

(Figure 4.4 D and E). Thus, while proteolytic degradation of inflammasome components may occur at later times following LMP, this cannot explain the rapid suppression of

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inflammasome assembly induced within several minutes after exposure to

supramillimolar LLME.

LMP-generated signals for activation of caspase-1-independent necrotic cell death

Lysosome-dependent necrosis is induced by extensive disruption of the lysosomal membrane and cytosolic accumulation of lysosomal cathepsins and other hydrolyases

(94). Cathepsin activity is required for LMP-induced cell death, as cell viability is restored upon treatment with the pharmacologic cathepsin inhibitor CA-074 or overexpression of cystatins (endogenous inhibitors of cathepsins) (94). However, the downstream signaling pathways leading to cell death subsequent to LMP remain uncharacterized. Our findings identified Ca2+ influx as one mechanism linking lysosome

destabilization and necrotic cell death. We found that withdrawal of extracellular Ca2+

markedly suppressed LMP-induced necrotic cell death as indicated by reduced LDH

release in BMDC treated with LLME in 0 Ca2+ medium (Figure 4.5 A). As summarized

in Figure 4.7, our findings indicate a dual role for influx of extracellular Ca2+ in the

response of BMDC to lysosome destabilization; increased cellular [Ca2+] inhibits NLRP3

inflammasome activation (and the subsequent processing/release of IL-1β) while promoting necrotic cell death in response to LMP.

Notably, a physiological role for LMP-induced cell death has been identified in the process of mammary gland involution. Upon cessation of lactation, STAT3 activity causes upregulated expression of cathepsins B and L, as well as decreased expression of the cathepsin inhibitor Spi2A (161). STAT3 activity also causes mammary epithelial cells to phagocytize Milk Fat Globules (MFGs) remaining in the mammary gland lumen.

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MFG degradation within lysosomes results in accumulation of oleic acid, which

permeabilizes the lysosomal membrane to causes release of lysosomal cathepsins into the

cytosol and necrotic death of mammary epithelial cells by an as yet unidentified

mechanism (95). It is relevant to consider whether the LMP-induced Ca2+-dependent cell death we have identified in myeloid leukocytes may be a general mechanism in other models of LMP-mediated cell death.

LLME is metabolized into oligomeric poly-leucine chains by the activity of cathepsin

C to mediate lysosome permeabilization and LMP-induced necrosis. Cathepsin C is most highly expressed by myeloid cells, NK cells, and T cells (113) rendering these cell types particularly susceptible to LLME-induced cell death. Indeed, early studies by Lipsky and colleagues extensively characterized the specificity and mechanisms for LLME-induced death of various human blood cell types to explore its possible application as an ablative or immunosuppressive chemotherapy (113,162-164). LLME was specifically evaluated as a therapy for treatment of graft vs. host disease (GVHD) following allogeneic bone marrow transplant (165-167). Human bone marrow was treated ex vivo with LLME in order to eliminate donor macrophages, DCs, NK cells, or CD8+ T cells that could react against recipient tissues. However, this LLME treatment was not effective in completely preventing GVHD.

Although NLRC4-mediated activation of caspase-1 dependent pyroptosis is a major mechanism for control of S. typhimurium (33), Lage et al. recently demonstrated that bacterial flagellin additionally engages a caspase-independent necrotic cell death pathway that contributes to clearance of S. typhimurium infection (168). That study used caspase-

1/11-/- macrophages to show that bacterial flagellin causes lysosome destabilization,

134 release of lysosome cathepsins into the cytosol and necrotic cell death with export of IL-

1α into the extracellular space. These findings demonstrate that cathepsin-mediated necrosis is an alternative mechanism for eliminating intracellular bacteria in the absence of caspase activation. Cathepsin-mediated necrosis may be the predominant mechanism for clearing S. typhimurium infection in B cells, where the bacterium downregulates

NLRC4 gene expression via phosphorylation of the Yap1 transcription factor (169).

In summary, the proposed non-linear signaling network (Figure 4.7) that integrates LMP with the gating of PM cation channels, NLRP3 inflammasome activation, and induction of inflammasome-dependent and inflammasome-independent modes of regulated myeloid cell death may be operative in multiple models of bacterial infection or sterile inflammation linked to lysosomal dysfunction. For example, both inflammasome signaling and necrotic macrophage death contribute to the development of atheromatous lesions in cardiovascular disease (112,170). Early atheroma are characterized by viable macrophages which internalize crystalline cholesterol and activate NLRP3 inflammasome signaling while advanced atheroma are defined by accumulation of macrophages undergoing necrotic cell death. Lysosome destabilization in response to accumulation of cholesterol crystals and/or destabilizing fatty acids may drive two distinct cellular responses, with low level LMP promoting inflammasome activation but high level LMP driving inflammasome-independent necrotic cell death.

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Chapter 5: Discussion and Future Directions

Increases in Extracellular [Ca2+] are not Required for NLRP3 Inflammasome Activation

in Response to K+ Efflux Agonists

Several recent studies have used pharmacologic approaches to implicate cytosolic

Ca2+ signaling in activation of the NLRP3 inflammasome(57,58). These reports have

utilized the intracellular Ca2+ chelator BAPTA and a variety of Ca2+ channel antagonists; these pharmacologic agents suppress inflammasome signaling in response to multiple

NLRP3 agonists. In the first part of this thesis (Chapter 3), I demonstrated that increases in cytosolic [Ca2+] are not required for NLRP3 inflammasome activation in response to two K+ efflux agonists: the bacterial ionophore nigericin (a K+/H+ exchanger that inserts

into cellular membranes) and extracellular ATP (which stimulates K+ efflux by opening

the P2X7 nonselective cation channel). Removal of extracellular Ca2+ or depletion of ER

Ca2+ stores did not diminish ASC oligomer formation, caspase-1 processing or IL-1β

processing/release in response to K+ efflux agonists. We also demonstrated that increases

in cytosolic [Ca2+] are not sufficient to activate the NLRP3 inflammasome on a timescale

of 30 min. In accordance with previously published findings, treatment of BMDC with

the Ca2+ ionophore ionomycin (which causes influx of extracellular Ca2+ and release of

ER Ca2+ stores) failed to induce caspase-1 processing or release of mature IL-1β. The

2+ 2+ Ca -mobilizing GPCR agonists R568 (synthetic agonist for the Gq-coupled Ca -sensing

receptor), submillimolar ATP or UTP (which activate the Gq/PLC-coupled P2Y2

receptors), and fMLP (a synthetic agonist of the Gi/PLC-coupled formyl peptide receptors) failed to cause inflammasome activation/IL-1β release within 30 min of

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stimulation (a timescale in which K+ efflux agonists induced robust inflammasome

activation). The intracellular Ca2+ chelator BAPTA (used to implicate increases in

cytosolic [Ca2+] in inflammasome activation by previous studies) suppressed nigericin-

induced caspase-1 processing/IL-1β release under conditions where no increases in

cytosolic [Ca2+] were observed (in the absence of extracellular Ca2+). Thus, the inhibitory

effect of BAPTA on inflammasome signaling is not due to its ability to chelate cytosolic

Ca2+. 2-APB is a pharmacologic agent previously reported to inhibit NLRP3 activation in

response to a variety of stimuli presumably via its inhibitory effect on release of ER Ca2+

2+ stores or influx of extracellular Ca into the cytosol through IP3R channels/Store-

Operated Ca2+ Entry channels, respectively. In accordance with these previous reports, we found that 2-APB potently inhibited the nigericin-induced NLRP3 inflammasome

signaling cascade. However, treatment of BMDC with 2-APB caused large increases in cytosolic [Ca2+]. These findings indicate that the suppressive effect of 2-APB on NLRP3

inflammasome activation does not result from its ability to inhibit increases in cytosolic

[Ca2+].

In summary, our findings from Chapter 3 indicate that a rapid decrease in

cytosolic [K+] is a highly efficacious signal for activation of NLRP3 inflammasome signaling, regardless of the presence/absence of an increase in cytosolic [Ca+ 2]. Influx of

extracellular Ca2+ into the cytosol or mobilization of ER Ca2+ stores is not sufficient to

cause rapid inflammasome activation. Our findings support the conclusion of Munoz-

Planillo et al. that a reduction in cytosolic [K+] is the critical signal for activation of the

NLRP3 inflammasome. However, the mechanism by which a decrease in [K+] is coupled

to the conformational activation of NLRP3 remains unknown. Moreover, changes in

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cytosolic [K+] may also act downstream of NLRP3 at the level of ASC aggregate formation. Given the proposed role for mitochondrial dysfunction in NLRP3 activation

(Chapter 1), perturbation of mitochondrial homeostasis by disruption of the normal

cytosolic [K+] is also a relevant area for future investigation.

2-APB Activates an Acid-Sensitive Channel in BMDC, Which May be Involved in the

Inflammatory Response to Extracellular Acidosis

An ongoing project in the lab is investigating the mechanism underlying 2-APB

induced Ca2+ influx in BMDC. Multiple studies have demonstrated the ability of 2-APB to act as an agonist of TRPV family channels (TRPV1, TRPV2, TRPV3)(135). We therefore hypothesized that 2-APB is activating a TRPV channel expressed on the plasma membrane of BMDC. Preincubation of BMDC with the noncompetitive pan-TRP channel inhibitor Ruthenium Red (RuRed) suppressed the 2-APB induced Ca2+ influx(171) (Figure 5.1 A). We also observed that the 2-APB induced Ca2+ influx was modulated by extracellular pH; incubation of BMDC in acidic basal salt solution (pH 6.5) enhanced channel opening in response to 2-APB (Figure 5.1 B). Previous studies have reported that TRPV1 and TRPV3 channels are sensitive to changes in pH. Acidic extracellular pH (6.0-7.0) potentiates TRPV1 opening in response to heat and the chemical ligand capsaicin(172). TRPV3 is activated by intracellular acidification

resulting from application of glycolic acid to the extracellular medium or application of

low pH solution to the cytosolic side of the plasma membrane by inside-out patch

clamping(173). There are currently conflicting reports in the literature regarding

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expression of TRPV1 in murine dendritic cells(174,175). To determine whether BMDC express functional TRPV1 channels, we utilized a pharmacologic approach and stimulated BMDC with two well-characterized TRPV1 agonists (capsaicin and resiniferatoxin [RTX]). If BMDC express TRPV1, we expected to see a large influx of extracellular Ca2+ into the cytosol upon application of these agonists. In accordance with

the study performed by O’Connell et al. (175), we did not observe an increase in

cytosolic [Ca2+] upon treatment of BMDC with TRPV1 agonists (Figure 5.1 C).

The above findings indicated that TRPV2 and TRPV3 are the best candidates for

the 2-APB activated channel in BMDC. A variety of 2-APB analogs are known to have

differential effects on the TRPV3 channel. In a study utilizing HEK293T cells expressing

recombinant TRPV3, Chung et al. observed that DPBA (a boron-containing analog of 2-

APB) also stimulated TRPV3 channel opening. On the other hand, DPTHF (an analog of

2-APB lacking the boron atom) was unable to open TRPV3(176). We found that treatment of BMDC with DPBA induced a large and rapid increase in cytosolic Ca2+,

while DPTHF was unable to stimulate Ca2+ influx (Figure 5.1 D). Chung at al. also found

that DPTHF inhibited 2-APB induced Ca2+ influx in TRPV3-expressing HEK293

cells(176). In accordance with these studies, we observed that pretreatment of BMDC

with DPTHF inhibited Ca2+ influx in response to 2-APB (Figure 5.1 E). As described

above, TRPV3 channel opening is potentiated by acidification of the cytosol. Nigericin is

a K+/H+ ionophore described in our previous studies. Upon insertion into the plasma

membrane, nigericin causes cytosolic acidification by transporting protons down their

concentration gradient from the extracellular space into the cytosol. We observed that

cytosolic acidification by pretreatment with nigericin potentiated the Ca2+ influx response

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to a subthreshold (30µM) concentration of 2-APB (Figure 5.1 F). These observations

provide further evidence that 2-APB activates TRPV3 channels in dendritic cells. There

are currently no studies in the literature examining TRPV3 protein expression in murine

dendritic cells. Studies are currently underway in our lab to detect TRPV3 expression in

BMDC by western blot. We will also perform surface biotinylation experiments to measure expression of TRPV3 on the surface of BMDC. We are also working to obtain

TRPV3-/- BMDC from collaborators; once we receive these cells, we will stimulate them

with 2-APB and compare the Ca2+ influx response to that observed in WT BMDC. We

expect that Ca2+ influx in response to 2-APB will be eliminated in TRPV3-/- BMDC.

Septic shock is the leading cause of death in US critical care units(177). Septic

shock is caused by infection and pro-inflammatory cytokines that are released in response

to pathogens. These pro-inflammatory mediators cause hypotension, heart failure, and

hypoperfusion of multiple organs(177). One of the major mechanisms driving septic

shock is opening of pyroptotic channels in response to activation of inflammatory

caspases (caspases 1, 4, 11) and the subsequent production of eicosanoids (including

prostaglandins and leukotrienes)(111,178). Inflammasome-dependent release of prostaglandins and leukotrienes results in leakage of vascular fluid and systemic hypoperfusion(111). Septic shock is also characterized by systemic lactic acidosis(179) and a recent study has demonstrated the ability of acidic extracellular pH to stimulate

NLRP3 inflammasome activation and pyroptosis in macrophages. Rajamaki et al. observed that exposure of macrophages to acidic extracellular medium caused caspase-1 processing over a time scale of several hours(180). Inflammasome activation was demonstrated to be dependent upon a gradual acidification of the cytosol caused by the

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Figure 5.1: Treatment of BMDC with 2-APB causes opening of an ion channel with

the pharmacologic profile of a TRPV family member. A) BMDC were preincubated in

the presence/absence of 1µM ruthenium red for 5 min. Cells were then stimulated with

100µM 2-APB at t=5 min. B) BMDC were transferred into basal salt solution (BSS) of

pH 7.4 or pH 6.5. Cells were incubated in BSS for 5 min and stimulated with 100µM 2-

APB at t=5 min. C) BMDC were stimulated with 100µM 2-APB, 14µM capsaicin or

5µM resiniferatoxin (RTX) at t=5 min. D) BMDC were stimulated with 100µM DPBA or 100µM DPTHF at t=5 min. E) BMDC were preincubated with 100µM DPTHF for 10 min starting at t=5 min. Cells were then stimulated with 100µM 2-APB at t=15 min. F)

BMDC were treated with 10µM NG at t=0 min followed by stimulation with 30µM 2-

APB at t=5 min.

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Figure 5.1

142 drop in extracellular pH. Treatment of macrophages with pharmacologic compounds that cause cytosolic acidification, including bafilomycin A (an inhibitor of the plasma membrane V-type H+ ATPase) and EIPA (an inhibitor of plasma membrane Na+/H+ exchangers), also stimulated NLRP3 inflammasome activation(180). As discussed above,

TRPV3 is a non-selective cation channel activated in response to acidification of the cytosol. We hypothesize that TRPV3 opening in response to cytosolic acidification mediates K+ efflux necessary for NLRP3 inflammasome activation in response to extracellular acidosis. Future experiments in our laboratory will test this hypothesis by treating macrophages/dendritic cells with TRP antagonists (RuRed, La3+, Gd3+) prior to exposure of cells to acidic pH. These experiments will determine whether inhibiting

TRPV3 channel opening reduces inflammasome activation/IL-1β release in response to acidosis. The results of these studies may contribute to the development of TRPV3 antagonists as a novel therapy for septic shock.

What are the Channels that Mediate K+ Efflux in Response to Lysosome

Permeabilization?

We found that treatment of BMDC with the lysosome destabilizing agent LLME caused rapid influx of Ca2+ and efflux of K+ within 5-10 min after application of stimulus.

Thus, destabilization of lysosomes causes rapid changes in plasma membrane cation permeability. Our study has demonstrated that the nonselective cation channel TRPM2 is involved in mediating part of the K+ efflux response to lysosome destabilization.

However, the K+ efflux response was not completely absent in TRPM2-/- BMDC treated with lysosome destabilizing agents (LLME or alum). These findings indicate that other

143

channels must be involved in mediating plasma membrane permeabilization in response

to compromise of lysosome integrity. We propose that other TRP family channels could

play a role in mediating the K+ efflux response to LMP. In order to test this hypothesis,

future studies in the lab will use electrophysiology to study the effect of the pan-TRP channel inhibitors RuRed, Gd3+, and La3+ on K+ efflux in response to treatment of

BMDC/BMDM with LLME or particulates. If other TRP channels mediate plasma

membrane permeabilization in response to LMP, we expect that K+ efflux in response to

LLME/particulates will be decreased in myeloid leukocytes treated with pharmacologic

pan-TRP inhibitors.

A recent study has demonstrated that lysozyme aggregates cause nonselective

permeabilization of the plasma membrane. Lysozyme is an enzyme found in abundance

within lysosomes of macrophages/dendritic cells(181). Demuro et al. found that lysozyme is capable of forming amyloid-like oligomers which permeabilize the plasma membrane to Ca2+(182). Indeed, several studies have demonstrated that amyloidogenic

proteins are capable of forming discrete nonselective pores within the plasma

membrane(183-185). In addition to causing opening of TRP channels, lysosome

destabilization may result in the release of lysozyme into the cytosol and the formation of

oligomeric lysozyme aggregates. These aggregates may then form nonselective pores in

the plasma membrane that are permeable to K+ ions and mediate part of the K+ efflux required for NLRP3 inflammasome activation. In accordance with this hypothesis, Gustot et al. recently demonstrated that treatment of THP-1 monocytes with lysozyme

aggregates causes NLRP3 inflammasome activation via a K+ efflux dependent

mechanism(186). Mina et al. have reported that Poloxamer 188 (a tri-unit polymer)

144 rescues plasma membrane damage caused by amyloidogenic proteins(187). Future experiments in the lab will investigate the role of lysozyme-mediated pore formation in

K+ efflux in response to LMP by treating BMDC with LLME/particulates in the presence/absence of Poloxamer 188. If lysozyme aggregates are involved in inflammasome activation in response to loss of lysosome integrity, Poloxamer 188 would be expected to inhibit K+ efflux and downstream inflammasome signaling/IL-1β release in response to LLME/particulates. Lysozyme knockout mice are commercially available; we will obtain BMDC from these mice and study the K+ efflux response to

LLME/particulates. If lysozyme is involved in mediating part of the increased plasma membrane permeability to cations after lysosome destabilization, we expect that lysozyme knockout BMDC will demonstrate decreased K+ efflux in response to treatment with LLME/particulates.

Is Cathepsin Protease Activity Required for Changes in Plasma Membrane Cation

Permeability in Response to Lysosome Destabilization?

As described in Chapter 1, previous studies have demonstrated that cathepsin protease activity is required for NLRP3 inflammasome activation in response to lysosome destabilizers. However, it remains unclear how cathepsin activity stimulates formation of inflammasome complexes. We propose that cathepsins released into the cytosol cause opening of nonselective plasma membrane cation channels by inducing proteolytic cleavage of channel gating domains. Once opened by protease activity, these nonselective cation channels may mediate the K+ efflux required to activate NLRP3. A similar mechanism has recently been demonstrated for the pannexin 1 ATP-release

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channel. The C-terminal tail of pannexin 1 acts to block the transmembrane pore of the

channel. Caspases proteolytically cleave off the C terminal tail, causing it to dissociate

from the rest of the channel and opening the transmembrane pore(188). Alternatively, cathepsin activity could generate second messengers or activate cytosolic enzymes (e.g. kinases) that may gate plasma membrane cation channels. In order to determine whether cathepsin activity is necessary for gating of TRPs or other nonselective cation channels, we will treat BMDC with particulates (alum or monosodium urate) in the presence/absence of pharmacologic cathepsin inhibitors (CA-074, zFA) and monitor changes in cellular [K+] using atomic absorption spectroscopy. These studies will be

performed in caspase-1/11-/- BMDC in order to eliminate cation flux occurring through pyroptotic pores and in the presence of 0mM extracellular Ca2+ to eliminate cation flux

resulting from LMP-induced necrosis. If cathepsin activity is required for opening of

plasma membrane channels in response to lysosome permeabilization, we expect that

cathepsin inhibitors will reduce K+ efflux in response to particulates.

What is the Mechanism by Which Complete Loss of Lysosome Integrity Inhibits NLRP3

Inflammasome Assembly?

In the second part of this thesis (Chapter 4), we set out to quantify the extent of

lysosome permeabilization required to activate the NLRP3 inflammasome. We identified

a biphasic relationship between the extent of LMP and the efficacy of NLRP3 activation

such that inflammasome signaling is stimulated by low-level LMP, but rapidly and

efficiently inhibited by massive lysosome disruption.

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We propose that there is an inhibitory factor released from permeabilized lysosomes that interferes with NLRP3 inflammasome activation or assembly. This factor does not cause degradation of inflammasome components (NLRP3, ASC, pro-caspase-1, pro-IL-1β). However, it exerts a “dominant negative” inhibitory effect on the inflammasome machinery and inhibits inflammasome assembly in response to the K+

efflux agonist nigericin. As described in Chapter 1, inflammasome components are

regulated at the post-translational level and the lysosomal inhibitory factor may interfere

with one or several of these post-translational modifications. NLRP3 is ubiquitinated in the basal state and must be deubiquitinated by BRCC3 in order to respond to “signal 2” activators(70). On the contrary, ASC must be linearly ubiquitinated by LUBAC in order to mediate inflammasome signaling(76). The lysosomal inhibitory factor may interfere with the function of one or both of these ubiquitin modifying enzyme complexes. The ubiquitination state of inflammasome components may be assayed by immunoprecipitation of NLRP3 or ASC in cell lysates with subsequent blotting against

K48, K63 or linear ubiquitin modifications. We expect that massive lysosome destabilization within BMDC will cause increased NLRP3 ubiquitination and reduced linear ubiquitination of ASC compared to that observed with submaximal LMP. ASC is

also regulated by the activity of kinases and phosphatases. In LPS-primed myeloid cells,

ASC is localized to the perinuclear space and associates with IKKα(73). ASC must be dephosphorylated by PP2A and dissociate from IKKα in order to form active inflammasome complexes (ASC specks)(73). The lysosome inhibitory factor may interfere with the dissociation of IKKα from ASC by inhibiting PP2A activity.

Interaction of ASC and IKKα may be assayed by immunoprecipitation of ASC-IKKα

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complexes. We expect that massive lysosome permeabilization within BMDC will cause

increased association of IKKα with ASC in the perinuclear space and an increased

amount of immunoprecipitated ASC-IKKα complexes compared to that observed in

response to submaximal LMP.

Are Inflammasome-Independent Cytokines Released by Myeloid Cells in Response to

Complete Lysosome Permeabilization?

In chapter 4 of this thesis, we demonstrated that treatment of BMDC with

supramillimolar concentrations of the lysosome destabilizer LLME does not result in the

efficient release of IL-1β despite causing complete permeabilization of lysosomes.

However, complete lysosome permeabilization may cause the release of other pro-

inflammatory cytokines from myeloid cells (such as IL-1α) into the extracellular

medium. Pro-IL-1α is expressed constitutively within the cytosol of myeloid cells.

However, myeloid pro-IL-1α is basally bound to intracellular IL-1R2 which prevents

cytokine processing by cytosolic calpain proteases(5). Active caspase-1 mediates the

proteolytic cleavage of intracellular IL-1R2, causing the receptor to dissociate from pro-

IL-1α. The cytokine is processed by calpains into the mature p17 form and released from

the cell; mature IL-1α then binds to IL-1R1 to mediate downstream signaling(5). Several

studies have proposed that IL-1α contributes to the inflammatory phenotype observed in

atherosclerosis and the formation of atherosclerotic plaques(189,190). Freigang et al.

observed that cholesterol and MSU crystals are capable of stimulating robust IL-1α release from macrophages even in the absence of significant inflammasome activation/IL-1β release(189). These findings bring up the interesting possibility that

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some of the lysosomal proteases released from permeabilized lysosomes may also be able

to cleave intracellular IL-1R2 and promote its dissociation from pro-IL-1α. As described

in chapter 4, treatment of BMDC with LLME causes substantial influx of extracellular

Ca2+ into the cytosol and calpain proteases are activated in response to elevated cytosolic

[Ca2+](191). Activated calpains could then process pro-IL-1α into its mature form, which

would then be released into the extracellular medium through the permeabilized plasma

membranes of cells undergoing LMP-induced necrosis. To test this hypothesis, we will treat BMDC with supramillimolar concentrations of LLME and measure release of IL-1α

by ELISA and western blot. We expect to see the release of substantial levels of mature

IL-1α in response to complete lysosome permeabilization.

Does Inflammasome Activation in Response to Lysosome Collapse Have a Role in

Maintenance of Tissue Homeostasis or the Response to Acute Tissue Injury?

Studies by our group and others have focused on the role of lysosome

destabilization in the dysregulated inflammatory signaling characteristic of crystal-

induced arthropathies, type 2 diabetes (lysosome destabilization induced by IAPP), or

metabolic syndrome (which involves destabilization of lysosomes by saturated fatty

acids) (Chapter 1). A question for further research is to determine whether activation of

inflammasome signaling by lysosomal destabilization plays a role in maintaining tissue

homeostasis in the absence of chronic inflammatory disease. A study by Yamasaki et al.

has proposed a role for lysosome destabilization-induced NLRP3 inflammasome

signaling in the repair of acute tissue injury(192). This group studied the inflammatory

response of macrophages to hyaluronan (HA), a large glycosaminoglycan that is a

149 constituent of the extracellular matrix and becomes fragmented upon tissue damage.

Binding of HA fragments to CD44 on the surface of macrophages triggers uptake of HA via endocytosis. HA accumulates within lysosomes and causes NLRP3 inflammasome activation/IL-1β release, likely by causing the collapse of lysosome integrity (although the authors did not directly examine the involvement of lysosome destabilization or cathepsin activity in HA-mediated inflammasome activation)(192). We will treat FITC- dextran loaded BMDM/BMDC with HA fragments and determine whether internalized

HA causes lysosome collapse using fluorescent microscopy. We expect that treatment of myeloid cells with HA will cause lysosome collapse, which will be visualized by the release of FITC-dextran from lysosomes into the cytosol. In order to test the contribution of cathepsin activity to inflammasome activation in response to HA, we will treat myeloid cells with HA fragments in the presence/absence of pharmacologic cathepsin inhibitors (CA-074, zFA). If cathepsin activity is required for inflammasome activation in response to HA, we expect that myeloid cells stimulated with HA in the presence of cathepsin inhibitors will demonstrate decreased caspase-1 processing and IL-1β processing/release compared to cells treated with HA in the absence of cathepsin inhibitors. HA-induced inflammasome activation and IL-1β release is likely to play an important role in the immune response to acute tissue damage by stimulating the recruitment of immune cells to the site of tissue injury and providing a supportive environment for these cells by promoting their survival/proliferation.

Ca2+ Influx in Response to Lysosome Destabilization Inhibits NLRP3 Inflammasome

Signaling and Promotes LMP-Induced Cell Death

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In Chapter 4 we demonstrate that LLME increases the permeability of the plasma

membrane to Ca2+, resulting in a large influx of extracellular Ca2+ into the cytosol within

5 min after addition of the LLME stimulus. Our results indicate that increases in cytosolic

[Ca2+] inhibit ASC filament formation, caspase-1 processing and IL-1β processing/release in response to lysosome destabilization. Increased cytosolic [Ca2+]

exerts an inhibitory effect at an upstream step in the inflammasome signaling cascade and

may interfere with either i) activation of the NLRP3 receptor, ii) association of

NLRP3/ASC, iii) the process of ASC filament assembly, or iv) causes cell death before

significant ASC aggregation has occurred.

Our findings also characterize influx of extracellular Ca2+ as an intermediate in

the signaling pathway linking lysosome destabilization and LMP-induced cell death.

Lysosome-dependent necrosis is known to be induced by extensive disruption of the lysosomal membrane, followed by translocation of lysosomal cathepsin proteases into the cytosol(94). Cathepsin activity is required for LMP-induced cell death, as cell viability is restored upon treatment with the pharmacologic cathepsin inhibitor CA-074 or overexpression of cystatins (endogenous inhibitors of cathepsins)(94). However, the signaling pathways leading to cell death subsequent to LMP remain uncharacterized. We found that influx of extracellular Ca2+ is a necessary event in the signaling cascade

linking lysosome destabilization to downstream cell death. We demonstrate that

withdrawal of extracellular Ca2+ potently suppresses necrotic cell death in response to lysosome destabilizers. In sum, our findings indicate a dual role for influx of extracellular

Ca2+ in the response of BMDC to lysosome destabilization; increased cellular [Ca2+]

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inhibits NLRP3 inflammasome activation (and the subsequent processing/release of IL-

1β) while promoting necrotic cell death in response to LMP.

Is There a Role for LMP-Induced Cell Death in the Response of Infected Cells to

Intracellular Bacteria?

We and others have studied the induction of cathepsin-mediated LMP-induced cell death in response to lysosome destabilization by lipotoxic agents, crystalline particulates, or small molecule lysosomotropic agents such as LLME(94). Recently,

LMP-induced cell death has been demonstrated to drive epithelial cell death during mammary gland involution(95). An unresolved issue in the field is whether LMP-induced necrotic cell death is also involved in the clearance of intracellular pathogens which inhabit protected endosome-derived compartments within host cells. One such pathogen is the bacterium Salmonella typhimurium, which infects myeloid/lymphoid cells and takes up residence in membrane-bound structures called Salmonella Containing Vacuoles

(SCVs)(193). S. typhimurium releases bacterial flagellin from SCVs into the cytosol and intracellular flagellin is detected by the NLRC4 inflammasome, which mediates bacterial clearance via caspase-1 dependent pyroptosis(33). Bacteria are released from pyroptotic myeloid cells into the extracellular space, where they are phagocytized by neutrophils and eliminated via ROS generated by the NADPH oxidase system(33). However, a report by Lage et al. has indicated that bacterial flagellin also engages a caspase-independent necrotic cell death pathway that contributes to clearance of S. typhimurium infection(168). This group observed in caspase-1/11-/- macrophages that bacterial

flagellin causes lysosome destabilization, release of lysosome cathepsins into the cytosol

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and necrotic cell death with export of IL-1α into the extracellular space. Use of pharmacologic cathepsin inhibitors abrogated cell death and IL-1α release in caspase-

1/11-/- BMDC. These findings demonstrate that cathepsin-mediated necrosis is an

alternative mechanism for eliminating intracellular bacteria in the absence of caspase

activation. Cathepsin-mediated necrosis may be the predominant mechanism for clearing

S. typhimurium infection in B cells, where the bacterium downregulates NLRC4 gene

expression via phosphorylation of the Yap1 transcription factor(169).

Contribution of Dissertation Research to the Field of Inflammasome Signaling

My studies have contributed to our understanding of the role of Ca2+ signaling in

NLRP3 inflammasome activation. Efficacious K+ efflux agonists (such as the bacterial

ionophore nigericin or activation of the P2X7 cation channel by extracellular ATP) do

not require increases in cytosolic [Ca2+] to activate NLRP3 inflammasome assembly.

Lysosome destabilizers are less efficacious than nigericin and ATP/P2X7 in causing efflux of cytosolic K+ and increases in cytosolic [Ca2+] negatively modulate NLRP3

inflammasome signaling in response to LMP. Additionally, influx of extracellular Ca2+

drives inflammasome-independent LMP-induced necrotic cell death. My studies have

also elucidated a unifying mechanism for NLRP3 activation in response to two distinct

categories of cellular damage: disruption of cytosolic cation homeostasis and disruption

of lysosome integrity. My thesis demonstrates that LMP rapidly alters the permeability of

the plasma membrane to K+ and Ca2+; efflux of cytosolic K+ then drives NLRP3

inflammasome assembly. Finally, my studies have provided a mechanism to explain

differential efficacy of lysosome destabilizers as NLRP3 inflammasome activators

153

observed by other studies. Low-level LMP is optimal for activation of NLRP3 inflammasome signaling. Massive permeabilization of lysosomes in myeloid cells causes the release of inhibitory factor(s) that suppress assembly of NLRP3 inflammasome components into active inflammasome complexes.

154

155

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