Trafficking of cholesterol to the ER is required for NLRP3 inflammasome activation.
- Altered cholesterol localization at the plasma membrane (PM) in Npc1−/− cells abrogated AKT–mTOR signaling by TLR4. However, the inability to activate the NLRP3 inflammasome was traced to perturbed cholesterol trafficking to the ER but not the PM. Accordingly, acute cholesterol depletion in the ER membranes by statins abrogated casp-1 activation and IL-1β secretion and ablated NLRP3 inflammasome assembly. By contrast, assembly and activation of the AIM2 inflammasome progressed unrestricted. Together, this study reveals ER sterol levels as a metabolic rheostat for the activation of the NLRP3 inflammasome.
Introduction
The
inflammasome is a multiprotein complex that plays critical roles in
infectious, inflammatory, and autoimmune diseases. The NLRP3
inflammasome is the most characterized inflammasome in terms of the
diverse stimuli that are known to activate it. Activation of the NLRP3
inflammasome requires assembly of NLRP3 and caspase-1 (casp-1) bridged
together through the adaptor protein ASC, wherein casp-1 undergoes
autoproteolytic processing. Subsequently, active casp-1 cleaves
precursor forms of cytokines interleukin (IL)–1β and IL-18, which can
then be secreted (Man and Kanneganti, 2015; Hamilton et al., 2017). Casp-1 also cleaves gasdermin D (GSDMD), making its N-terminal pore-forming domain active, leading to cell rupture (Kayagaki et al., 2015; Shi et al., 2015).
Distinct exogenous, endogenous, and environmental stimuli are known to
activate the NLRP3 inflammasome, implying that these stimuli do not bind
NLRP3 directly but likely converge on shared upstream pathways. The
mechanistic details of NLRP3 activation remain ambiguous.
Lipids are known to carry out diverse functions within cells, including
being a major component of cell membranes, and as signaling messengers.
Cholesterol is an essential lipid in mammalian cell membranes aiding
varied functions, the most fundamental of which are membrane integrity
and fluidity (Maxfield and Tabas, 2005).
Levels of cholesterol in the cell are maintained through de novo
synthesis in the ER, and uptake of low-density lipoproteins (LDLs)
derived from dietary cholesterol. Excess free cholesterol can be toxic
to cells; thus, sterol homeostasis needs to be integrated by a
combination of cholesterol uptake, biosynthesis, and efflux programs. At
the subcellular level, cholesterol follows an intricate pathway in
cells (Ikonen, 2008).
Exogenously obtained LDL bound to LDL receptor is internalized at the
plasma membrane (PM) and is transported through the endocytic pathway to
the late endosomes–lysosomes, where cholesterol esters within the LDL
core are hydrolyzed by acid lipases. Unesterified or free cholesterol
translocates through the lysosomal cholesterol transporter Niemann-Pick
C1 (NPC1) to other cellular sites such as the PM and the ER. In the ER,
cholesterol can be reesterified, permitting cytoplasmic storage in the
form of lipid droplets.
Until recently, cholesterol has mostly been accepted to have an
influence on immunity during pathological conditions such as in
atherosclerosis (Fessler, 2016).
However, evidence suggests that homeostatic lipid metabolism and
trafficking directly regulate the inflammatory pathways in macrophages.
For example, defective lipid trafficking in the absence of NPC1 leads to
the lysosomal storage disorder Niemann-Pick disease (Platt et al., 2012). Mutations in the cholesterol efflux transporter, ABCA1, give rise to signs and symptoms of Tangier disease (Fasano et al., 2012).
Similarly, perturbations in lipid metabolism contribute to several
human pathologies including cardiovascular, obesity, and
neurodegenerative diseases (Maxfield and Tabas, 2005).
In addition to contributing to the pathogenesis of several diseases,
cholesterol is also exploited by pathogens for their entry and
proliferation within host cells. Several pathogens that lack the
capacity for de novo sterol synthesis use cholesterol for their survival
and replication by either increasing host lipid biosynthesis or
redirecting cholesterol transport pathways (Coppens et al., 2000; Lauer et al., 2000; Carabeo et al., 2003; Kaul et al., 2004; Ilnytska et al., 2013).
These studies suggest that reducing lipid synthesis may serve to limit
nutrients available to pathogens, thus benefitting host cells.
Conversely, host cells need lipids for mounting a robust immune response
to infection through conserved pattern recognition receptors (Castrillo et al., 2003; York et al., 2015).
Together, these studies lead to the hypothesis that lipid homeostasis
is critical for an effective inflammatory response with implications for
homeostatic lipid trafficking in both infectious and inflammatory
diseases. Whether perturbations in homeostatic cholesterol trafficking
pathway impact inflammasome activation remains unknown.
In this study, by using pharmacological and genetic tools, we
demonstrate that selective perturbation of the cellular cholesterol
trafficking in macrophages ablates inflammasome activation.
Mechanistically, perturbed sterol trafficking in Npc1
deficiency leads to two distinct effects: altered PM cholesterol levels
resulted in inhibition of the AKT–mTOR pathway, while reduced
cholesterol trafficking to the ER blunted NLRP3 inflammasome assembly.
Accordingly, acute cholesterol depletion in the ER by statins decreased
IL-1β secretion, which could be restored by supplementing with exogenous
cholesterol. Our findings thus implicate sterol synthesis and
distribution as critical factors influencing the activation of the
inflammasome, thereby coupling lipid homeostasis to innate immune
signaling.
---
Activation of inflammasome is characterized by cell swelling and subsequent osmotic lysis in the form of pyroptotic cell death following GSDMD cleavage (Kayagaki et al., 2015; Shi et al., 2015). In control cells primed with either LPS or Pam3, stimulation with ATP caused pyroptotic cell death. However, upon exposure to U18666a, cell death, and thus the secreted levels of cytosol-localized cell death marker, lactate dehydrogenase (LDH), were markedly diminished (Fig. 1, G–I). Overall these results demonstrate reduced casp-1 activation, IL-1β and IL-18 secretion, and decreased pyroptosis when cellular cholesterol trafficking is inhibited.
...
We next performed experiments to determine the role of priming in our
assays.
Activation of the NLRP3 inflammasome requires two signals.
(1) The
first signal, which potentiates the NLRP3 inflammasome, is provided upon
Toll-like receptor (TLR) ligation and results in the synthesis of
pro–IL-1β and up-regulation of NLRP3 expression (Anand et al., 2011a). This first signal (also known as the priming step) is dependent on the transcription factor NF-κB (Bauernfeind et al., 2009; Hornung and Latz, 2010).
(2) The second signal, which is more robustly provided by purinergic receptor P2X7 agonist ATP, results in the assembly of NLRP3, casp-1, and adaptor protein ASC to form a multiprotein complex (Pelegrin and Surprenant, 2006).
...
A diverse array of signals is known to activate the NLRP3 inflammasome.
In contrast, ligands that activate the NLRC4 and AIM2 inflammasomes are
well defined.
The NLRC4 inflammasome is triggered by the recognition of
cytosolic flagellin and components of the bacterial secretion system by
upstream NAIP family members (Amer et al., 2006; Kofoed and Vance, 2011; Zhao et al., 2011).
AIM2 inflammasome, in contrast, is activated by recognition of double-stranded DNA of ∼80 bp in length (Jin et al., 2012).
Cholesterol supplementation restores inflammasome activation
Variation
in the levels of cholesterol and other lipids within the cell are
sensed by master transcriptional regulators SREBP1 and SREBP2, which,
respectively regulate fatty acid and cholesterol biosynthesis (Horton et al., 2002).
A feedback mechanism tightly regulates cholesterol de novo biosynthesis
to maintain homeostatic levels. Cholesterol depletion triggers ER
resident sterol cargo protein SCAP to escort SREBP2 to the Golgi, where
the precursor is cleaved to activate cholesterogenic genes. By contrast,
in cholesterol-replete cells, conformational change in SCAP promotes
insulin-induced gene–mediated retention of SREBP2 in the ER.
Picture:
Picture:
---
F) Homeostatic cellular cholesterol trafficking to the
ER is required for optimal inflammasome activation. Exogenously obtained
LDL bound to LDL-R is endocytosed at the PM. Cholesterol efflux from
the late endosome–lysosome compartment to other cellular sites such as
the PM and the ER is dependent on NPC1. (G) Blockade of
the cholesterol transporter NPC1 leads to lysosomal cholesterol
accumulation and a subsequent decrease in cholesterol pool in both the
ER and the PM. This in turn leads to decreased phosphorylation of AKT
and mTOR following TLR ligation, causing a reduction in SREBP2-dependent
lipogenesis. Additionally, perturbation in the ER cholesterol levels
leads to decreased association of NLRP3 and ASC, resulting in reduced
active casp-1 levels and IL-1β secretion. Furthermore, inhibition of ER
cholesterol levels by statins similarly blunts NLRP3 inflammasome
activation. LDL-R, low-density lipoprotein receptor; mTOR, mammalian
target of rapamycin; SREBP2, sterol regulatory element binding protein
2.
