Inflammasomes in non-immune cells: functions and role in disease
Author: Casper Berger, University of Utrecht, master student Infection and Immunity.
Daily supervisor: MSc. Lieneke Bouwman,, University Utrecht, Fac. of Veterinary Medicine, Dept. of
Infectious Diseases and Immunology.
Examiner: Prof. Jos van Putten, University of Utrecht, Fac. of Veterinary Medicine, Dept. of Infectious Diseases
and Immunology.
Second reviewer: Dr. Hélène Verheije, University of Utrecht, Fac. of Veterinary Medicine, Dept. of
Pathobiology
Abstract
Inflammasomes are intracellular protein complexes that consist of a molecular sensor, an ASC
adaptor protein and the cysteine protease caspase-1. Inflammasomes are involved in cleaving pro-IL1β and pro-IL-18 into their active forms. Numerous studies show that beside immune cells, nonimmune cells can express functional NLRP1, NLRP3, NLRC4, NLRP6, AIM-2, RIG-I and IFI16
inflammasomes. The function of inflammasomes in non-immune cells is still largely unknown, but the
inflammasome may be involved in several diseases including infections by pathogens, allergic
reactions, age-related macular degeneration, ischemia and reperfusion injury, diabetes and cancer.
Whether inflammasome activation is beneficial or detrimental to the development of these diseases is
often still unclear. This review gives an overview of the characteristics of inflammasomes in nonimmune cells and discusses their function in comparison to inflammasomes in immune cells.
Table of contents
Abstract ............................................................................................................................................ 1
1.
Inflammasome structure, function and activation ............................................................. 2
Structure of inflammasomes .......................................................................................................... 2
Functions of inflammasomes ......................................................................................................... 4
Mechanisms of inflammasome activation ...................................................................................... 5
Regulation of inflammasome activity ............................................................................................ 5
2.
Pathogen and microbiota induced inflammasome formation in non-immune cells........ 6
Pathogens ....................................................................................................................................... 6
Microbiota ...................................................................................................................................... 7
3.
(Hyper) response to foreign biological molecules ............................................................... 8
4.
Response to endogenous proteins, metabolites and danger signals .................................. 9
Hyperhomocysteinemia and psoriasis ............................................................................................ 9
Glucose and the NLRP3 inflammasome ........................................................................................ 9
The NLRP3 inflammasome and age-related macular degeneration ............................................. 10
Ischemia and reperfusion injury ................................................................................................... 11
Atherosclerosis ............................................................................................................................. 11
5.
Response to non-biological substances and stimuli .......................................................... 12
6.
Inflammasomes in wound healing, tumor development and pain sensitivity ................ 13
Wound healing and tumors .......................................................................................................... 13
1
Pain sensitivity ............................................................................................................................. 14
Overview of inflammasome types in non-immune cells ............................................................. 16
7.
Discussion............................................................................................................................. 18
References ....................................................................................................................................... 19
1. Inflammasome structure, function and activation
Inflammasomes are intracellular protein complexes involved in the activation of inflammatory
cytokines in response to pathogens and danger signals, resulting in inflammation and/or cell death.
This makes inflammasomes important players in the innate immune response. The primary focus of
studies on inflammasomes so far has been their role in dedicated immune cells of hematopoietic
origin. However, the molecular components of inflammasomes are also present in non-immune cells.
This review focuses on the role of inflammasomes in non-immune cells. In the first section an
introduction on the inflammasome structure and the different inflammasome types is provided. The
second part gives an overview of the roles of inflammasomes in diverse processes in non-immune
cells including the response against pathogens, ischemia and reperfusion injury, metabolic disorders,
auto-immune diseases, allergic reactions and cancer. Finally I will discuss the relative role of
inflammasomes in non-immune cells compared to immune cells. An overview of studies on
inflammasomes in non-immune cells is provided in Table 1.
Structure of inflammasomes
The primary function of inflammasomes in immune cells is to trigger inflammation and cell death in
response to pathogen associated molecular patterns (PAMPs) and danger associated molecular
patterns (DAMPs). This is primarily mediated through the processing of pro-IL-1β and pro-IL-18 into
their inflammatory active form. Canonical inflammasomes consist of three components to accomplish
this: a sensor molecule, the adaptor protein apoptosis-associated speck-like protein (ASC) and the
cysteine protease caspase-1 (Figure 1). The sensor molecule binds to adaptor protein ASC with its
pyrin domain when activated by PAMPs and DAMPs. ASC in turn binds monomeric pro-caspase-1
via its caspase recruitment domain (CARD). This results in auto-cleavage of pro-caspase-1 into the
active caspase-1 which subsequently forms heterotetramers of the P10 and P20 caspase- subunits. The
caspase-1 in the protein complex then cleaves the interleukins pro-IL-1β and pro-IL-18, resulting in
release in their activated form[1].
Molecular sensor
ASC
P20
pro-IL1-β
1β
2
P10
IL1-1β
Figure 1. Schematic overview
of inflammasome formation.
A molecular sensor is directly
or indirectly activated by
PAMPs and DAMPs,
facilitating subsequently
binding to the adaptor protein
ASC. ASC then binds procaspase-1 which results in
auto-cleavage into the P10 and
P20 subunit. Heterotetramers
of the P10 and P20 subunit are
formed which cleave pro-IL1β and pro-IL-18.
Different inflammasomes can be formed depending on the type of molecular sensor that is involved.
Many of the molecular sensors are NOD like-receptors (NLRs). Most NLRs involved in
inflammasome formation including NLRP3, NLRP6 and NLRP7 contain a pyrin domain with which
they can bind to ASC. However, some NLRs lack a pyrin domain. NLRC4 contains a CARD instead
of pyrin domain and can directly interact with caspase-1. Although ASC is not required for pro-IL-1β
processing, it does enhance it[1]. Human NLRP1 contains both a pyrin and a CARD domain whereas
murine NLRP1-a and –b (mice have three NLRP1 isoforms) only contains a CARD domain. NLRP1c
has a truncated CARD domain[2][3]. Although human NLRP1 has a CARD domain and can therefore
directly interact with pro-caspase-1, ASC has been shown to enhance NLRP1 inflammasome activity
in humans[2]. All NLRs also contain a NAIP, CIITA, HET-E and TP1 (NACHT) domain which is
involved in ATP-dependent oligomerization with other NACHT domain containing proteins,
facilitating the complex formation. They also have C-terminal leucine-rich repeats which have
regulatory functions[1].The non-NLR proteins retinoic acid-inducible gene I (RIG-I), interferon
gamma-inducible protein 16 (IFI16) and absent in melanoma-2 (AIM-2) can also act as molecular
sensors and form inflammasomes[1][4]. IFI16 and AIM-2 contain one or more HIN domains which
bind double stranded DNA[1]. RIG-I can also bind to double stranded DNA[4]. Off all the known
inflammasome types, the NLRP3 inflammasome is the best characterized. An overview of
inflammasome formation is provided in Figure 2.
Figure 2. Schematic overview of NLRP1, NLRP3, NLRC4 (IPAF) and AIM-2 inflammasomes.
NLRP1 and NLRC4 contain a CARD domain and can directly interact with caspase-1 (although
ASC enhances NLRP1 and NLRC4 inflammasome activity[1][2]). NLRP3and AIM2 bind ASC
with their pyrin domain which in turn binds to caspase-1 via its CARD domain. Binding of the
molecular sensor directly or via ASC to caspase-1 results in caspase-1 auto-cleavage yielding P20
and P10 subunits (cleavage sites indicated with arrows). These can form heterotetramers which
can cleave pro-IL-1β. Image originally published by K Schroder and J Tschopp 2010[2].
Variations in inflammasome components are not limited to the sensor proteins, but also exist in the
ASC protein and the protease. Three different isoforms of ASC are known to exist (ASC –a, - b and –
c). ASC-b lacks the linker domain between the pyrin and CARD domain. ASC-b has been shown in
vitro to be able to form functional NLRP3 inflammasomes while ASC-c lacks parts of the pyrin
domain but still has a functional CARD domain. ASC-c has been shown in vitro to inhibit
inflammasome formation, possibly by competing for binding sites on pro-caspase-1 with conventional
full-length ASC-a[5]. ASC has also been reported to have tumor repressive effects independent from
inflammasomes by activating P53[6].
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Activation of pro-caspase-1 auto cleavage can also occur through murine caspase-11 (caspase-4
and/or 5 in humans). Caspase-11 may be activated by TLR4 and the downstream TRIF pathway.
Activation of this pathway results in interferon -α and -β release, which results in pro-caspase-11 auto
cleavage and subsequent pro-caspase-1 auto-cleavage of the NLRP3 inflammasome. Pro-caspase-11
is also auto-cleaved when expressed at sufficiently high cellular levels[7]. The mechanism by which
caspase-11 affects NLRP3 inflammasome activation is not fully understood[8].
Functions of inflammasomes
The primary function of inflammasomes appears the processing of pro-IL-1β and pro-IL-18 and their
subsequent release by non-classical protein secretion (because IL-1β and pro-IL-18 lack a signaling
peptide). IL-1β and IL-18 promote inflammation by recruiting immune cells, inducing fever,
increasing pain sensitivity and vasodilation[9]. It should be noted that IL-1β has been reported to be
secreted in extracellular membrane vesicles[10] beside being released as a soluble proteins. Proteins
present in extracellular vesicles cannot be detected with assays such as ELISA unless the vesicles are
lysed and may therefore be missed by assays.
Inflammasome-independent cleaving of pro-IL-1β, pro-IL-1β can also occur by a protein complex
that consists of mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALTI1),
ASC and caspase-8 can also process pro-IL-1β. This complex can be activated by specific pattern
recognition receptors such as dectin 1, the ripoptosome (a protein complex involved in cell death in
response to genotoxic stress) and activation of the tumor necrosis factor receptor (TNFR) CD95[1].
Beside IL-1β, the interleukin IL-1α, which also binds the IL-1 receptor, can trigger inflammation.
Whether IL-1α processing and release are also controlled by inflammasomes is less clear. The fulllength IL-1α protein can be cleaved by a calpain-like protease, but unlike IL-1β both the cleaved and
the non-cleaved form are biologically active. IL-1α cleavage and release may be partially dependent
on the inflammasome and caspase-1 (but not the protolithic activity of caspase-1). IL-1α may not be
actively released at all and rather act as an inflammatory signal when it leaks from cell dying due to
non-apoptotic cell death[11]. However, Lee et al. 2009 report that a caspase-1 inhibitor reduced IL-1α
release from the epidermis[12].
Inflammasomes are also involved in pyroptosis. Pyroptosis is a programmed cell death pathway
distinct from apoptosis and dependent on caspase-1. Pyroptotic cells swell resulting in rupture of the
plasma membrane and the release of inflammation inducing molecules. Caspase-1 and caspase-11 are
involved in pyroptosis and inflammasome formation can result in pyroptosis. Multiple inflammasome
types such as NLRP3, NLRC4 and AIM-2 can initiate pyroptosis[9][13].
In addition to interleukin processing and pyroptosis, inflammasomes may have additional
functions which are relatively unexplored. One study identifies 41 substrates of caspase-1 by using a
diagonal 2D gel with in-gel caspase-1 digestion followed by identification of digested proteins with
mass spectrometry. The identified substrates are involved in diverse processes including the
cytoskeleton and energy metabolism. Five of the identified substrates are cytosolic enzymes in the
glycolysis pathway. Some of these enzymes are cleaved in mouse macrophages in response to
Salmonella infection in vitro and this is dependent on caspase-1. Additionally, lactate levels are
higher in Salmonella infected macrophages isolated from caspase-1 knockout mice compared to
macrophages isolated from wildtype mice. This may suggest that caspase-1 activation reduces
glycolysis[14].
In a different study, caspase-1 activation by pore forming toxins results in a ASC, NLRP3 and
NLRC4 -dependent sterol regulatory element binding protein -1 and -2(SREBP) activation which in
turn results in increased cell survival of HeLa cells[15]. SREBPs are transcription factors that play a
central role in membrane biogenesis. How caspase-1 activates SREBPs has not been established.
Interestingly, SREBP-1a has also been shown to directly stimulate NLRP1a transcription but not of
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NLRP1c[16]. These studies show that caspase-1 has more functions beside the processing of pro-IL1β and pro-IL-18 and pyroptosis. They also indicate that inflammasome activation may have more
diverse functions than interleukin processing and pyroptosis.
Mechanisms of inflammasome activation
The sensor molecules of inflammasomes can directly detect distinct signals such as the bacterial cell
wall component muramyl dipeptide by NLRP1 and double stranded DNA by AIM-2. The NLRP3
inflammasome on the other hand can be activated by a wide range of stimuli including pathogens,
metabolites, many inorganic crystalline compounds and UVB. Several reports suggest that an efflux
of K+ is a central mechanism in NLRP3 inflammasome formation and may help explain the broad
specificity. An efflux of K+ has been shown to result in reactive oxygen species (ROS) formation and
subsequent NLRP3 inflammasome dependent caspase-1 activation[17]. Permeabilization of the
plasma membrane by pore forming bacterial toxins also results in a K+ efflux and subsequent
inflammasome activation[15]. Additionally, the phagocytosis of crystalline compounds such as urate
crystals can result in ROS formation by damaging lysosomes resulting in NLRP3-inflammasome
formation[2]. A more recent study suggests that although a K+ efflux is essential for NLRP3
inflammasome activation, ROS formation is not critical to the process[18]. The leaking of lysosomal
enzymes into the cytosol such as cathepsin B has also been implicated in NLRP3 inflammasome
activation[19].
Beside inflammasome activation, upregulation of pro-IL-1β is also required. Pro-IL-1β is not
constitutively expressed in most cell types but instead is upregulated by NF-κB in response to NOD
and Toll-like receptor activation.
Regulation of inflammasome activity
The activity of the inflammasome is controlled at multiple levels. Beside the three core-components
described above the formation of some inflammasomes may require the involvement of co-receptors.
NLRC4 may require NAIP family members for inflammasome formation. NAIP family members can
detect proteins of pathogens such as flagellin by NAIP5 and NAIP6 and a type III secretion system
component is detected by NAIP2 in mice. Only a single NAIP orthologue is known in humans
though.
The formation of the NLRP1inflammasome is likely dependent on NOD2[1][2]. For NLRP3,
activity is controlled at both the transcriptional and protein level. NLRP3 expression is low under
normal conditions and needs to be upregulated to increase inflammasome formation. Additionally,
deubiquitination of NLRP3 by the deubiquitinase BRRC3 enhances NLRP3 activity[1]. Autophagy
may play a role in limiting inflammasome activation by preventing accumulation of factors that
stimulate inflammasome formation, such as ROS and mitochondrial DNA in the cytoplasm[20].
Negative regulation of NLRP3 inflammasomes has been shown to occur by direct contact of CD4+ T
cells and is possibly dependent on a CD40 ligand[1][2]. Many more factors that regulate
inflammasome formation have been described but it is outside the scope of this review to describe
them all. An excellent in-debt review on regulation of inflammasomes has been written by Latz et al.
2013[1].
Inflammasomes and their function appear to be more complex as originally described. More sensor
molecules continue to be identified and some can also directly bind to pro-caspase-1 rather than to
ASC. Inflammasomes components are regulated at multiple levels by a variety of factors. Alternative
inflammasome activation pathways have been identified and inflammasome independent processing
of IL-1β can also occur. Regarding their functions, beside their well-known role in interleukin
processing and pyroptosis, inflammasomes have been shown to affect membrane biogenesis and a
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large number of substrates have been identified for caspase-1 including glycolysis enzymes. These
additional levels of complexity may help to explain some of the observed discrepancies between
different studies and underline the importance of the more biologically relevant in vivo models
compared to in vitro models to verify findings on inflammasomes.
They also make it more difficult to define what an inflammasome is and what is not. ASC is not
strictly required and neither is interleukin processing its only function. Pro-caspase-1 activation, a
sensor molecule that recognizes PAMPs and/or DAMPs and the formation of a protein complex
appear indispensable for canonical inflammasomes. However, caspase-8 dependent IL-1β processing
can also occur. This complex shares many properties with canonical inflammasomes and can
therefore arguably be termed a type of inflammasome as well. What both complexes do have in
common is that they are activated by stimulation of PAMP- and DAMP-sensing molecules, form a
large protein complex that results in the activation of caspase-1 or caspase-8 which in turn can cleave
pro-interleukins to activate them.
2. Pathogen and microbiota induced inflammasome formation in non-immune cells
Many pathogen components are directly or indirectly recognized by the molecular sensors, resulting
in inflammasome activation and interleukin processing. The majority of studies focus on this role in
immune cells, but some studies also show inflammasome activation in response to pathogens in nonimmune cells. Beside recognizing pathogens, effects of inflammasomes on the composition of the
microbiota and resulting pathologies have also been reported[21][22].
Pathogens
One pathogen that has been shown to trigger inflammasome activation in non-immune cells is the
influenza A virus. Infection by influenza A virus results in a RIG-I and NLRP3 inflammasome
dependent IL-1β release by primary human lung epithelial cells in vitro[4]. RIG-I activation also
resulted in RIG-I, TLR3 and NLRP3 upregulation. This upregulation depends on interferon-β and the
type I interferon receptor 1 α chain (IFNAR1), which increases RIG-1, TLR3 and NLRP3
expression[4]. These results show the direct formation of NLRP3 and RIG-I inflammasomes in
response to a virus and an indirect role for RIG-I in NLRP3 inflammasome activation in non-immune
cells. The authors did not compare the IL-1β release to the amount of IL-1β released by macrophages
or other immune cells from the same donors, so the relative contribution to IL-1β release remains
currently unknown.
IFI16 inflammasome activation has also been implicated in response to Kaposi's sarcomaassociated herpesvirus (KSHV)[10]. Increased caspase-1 activation and processing of IL-1β were
observed in latently infected endothelial telomerase-immortalized human umbilical cells (TIVE) and
B-cells but not in uninfected cells. Co-immunoprecipation experiments showed that IFI16 forms
protein complexes with ASC and caspase-1. Fluorescence microscopy revealed that in non-infected
cells IFI16 is mostly localized in the nucleus but localizes mostly perinuclear with ASC during KSHV
infection. FISH staining of the KSHV genome shows that it colocalizes with IFI16, suggesting that it
is directly detected by IFI16[10]. KSHV normally infects B-cells in vivo so it remains to be seen
whether KSHV also results in inflammasome activation in non-immune cells in vivo. Yersinia
enterocolitica infection also triggers inflammasome activation[23]. Y. enterocolitica infection of
Caco-2 cells results in NLRP3 inflammasome-dependent IL-18 release triggered by a K+ flux. No IL1β release was observed however. Binding of the bacterial adhesin invasin (Inv) to α5β1 integrin
resulted in increased IL-18 release during infection due to increased IL-18 expression and is therefore
an alternative priming signal[23].
Inflammasome activation in non-immune cells in response to pathogens is not necessarily
beneficial for the host. An in vitro study shows that Chlamydia trachomatis triggers NLRP3
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inflammasome formation in HeLa cells[17]. The type III secretion system is required for this
activation. The type III secretion system triggers a K+ efflux which results in ROS formation and
subsequent NLRP3 inflammasome formation and caspase-1 activation. Interestingly, C. trachomatis
benefits from the observed inflammasome activation since inhibition of caspase-1 results in decreased
infection[17].
The bacterium Pseudomonas aeruginosa does not appear to stimulate inflammasome activation in
non-immune cells whereas it does in immune cells. One study observed hardly or no caspase-1
activation and IL-1β release of 4 different lung epithelial cell lines in response to P. aeruginosa
whereas blood mononuclear cells did show caspase-1 activation and IL-1β release[24]. Another study
compared IL-1β release from macrophages and the total lung tissue in response to P. aeruginosa[25].
They observed that the majority of IL-1β is released by macrophages and is caused by activation of
the NLRC4 inflammasome by the bacterial flagellin. Interestingly, inflammasome activation was
associated with more severe disease outcome in mice. Mice challenged with wildtype or a mutant P.
aeruginosa with nonfunctional flagella showed decreased survival and more tissue damage compared
to infection with a mutant P. aeruginosa that lack flagellin[25].
Microbiota
Two studies using knockout mice show that the NLRP3 and NLRP6 inflammasomes activity in nonimmune cells have an effect on the microbiota[21][22]. The changes in microbiota resulted in
inflammatory pathology. In one study, NLRP6, ASC and caspase-1 knockout and wt mice were given
dextran sodium sulfate (DSS) which is known to result in inflammation of the colon (colitis). The
knockout mice developed more severe colitis compared to wt mice, indicating a protective role of
NLRP6 inflammasomes. This phenotype was transferable to co-housed wt mice and treatment with
antibiotics reversed the more severe colitis of the knockout phenotype, suggesting that the phenotype
is caused by changes in microbiota caused by deficient inflammasomes. This effect was caused by IL18 rather than IL-1β as shown by knockout experiments. The changes in microbiota were primarily
caused by the NLRP6 inflammasome in non-immune cells as shown by knockout experiments with
adoptive transfer of IL-18 and NLRP6 knockout bone marrow[22].
Another study with very similar experiments indicates that the NLRP3 inflammasome likely plays
a role in non-alcoholic steatopepatitis (NASH). ASC, caspase-1 and NLRP3 knockout mice fed a
methionine-choline-deficient diet (which is known to induce NASH) have increased NASH symptoms
compared to wt mice. The more severe NASH phenotype was shown to be caused by changes in the
microbiota with co-housing experiments and antibiotic treatment. Adoptive bone-marrow experiments
confirmed that inflammasome activation in non-immune cells is responsible for the altered NASH
phenotype. The altered microbiota results in an increase of TLR4 and TLR9 agonists reaching the
liver through the portal vein, which triggers inflammation in the liver[21]. However, it was not
investigated how the altered microbiota results in more TLR4 and TLR9 ligands reaching the liver
through the portal vein.
Activation of NLRP6 inflammasome in goblet cells and epithelial cells has also been shown to
reduce mucus secretion[26]. This effect was caused by NLRP6 inflammasome induced reduction of
autophagy which in turn results in deficient mucus secretion and accumulation of mucus-containing
vesicles on the apical side of goblet cells[26].
Collectively, these studies show that inflammasomes in non-immune cells can have a role in response
to pathogens and commensal microbiota. The relative contribution of inflammasomes in non-immune
cells to the response against pathogens is difficult to determine. The variable activation of
inflammasomes observed in non-immune cells in response to different pathogens [25][24][4][17] may
indicate that inflammasome activation in non-immune cells is dependent on a number of factors such
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as the host immune system and the type of pathogen. Although IL-1β release of immune cells seems
higher than non-immune cells, the IL-β release by non-immune cells may still play a role in initiating
inflammation by recruiting and activating innate immune cells which can then further amplify the
immune response. The relatively much greater number of non-immune cells compared to the number
of immune cells in peripheral tissues should also be considered. Autocrine activation of surrounding
epithelial tissue may amplify the immune response. Epithelial cells are also the first cells to be
exposed to a pathogen which would make them good candidates to initiate an immune response.
Apart from interleukin processing and release, alternative functions of inflammasome activation in
non-immune cells should be considered such as pyroptosis and alterations in metabolism. These may
have a more prevalent role than interleukin processing in non-immune cells.
3. (Hyper) response to foreign biological molecules
Inflammasome activation in non-immune cells can also occur in response to non-pathogen derived
foreign biological molecules. Several studies implicate a role for NLRP3 and AIM-2 inflammasomes
in keratinocytes in the development of allergic skin responses. ASC, NLRP3 and IL-1R knockout
mice sensitized with dinitro-1-fluorobenzene (DNFB) or trinitrochlorobenzene (TNCB) on the skin
and later -re-exposed to the same compound showed reduced inflammation compared to wildtype in
the early phase of inflammation, and also in the late phase in IL-1R knockout mice[28]. Furthermore,
it was demonstrated that in vitro mouse keratinocytes release IL-1β in response to various stimuli
including sodium dedecyl sulfate (SDS), UVB (UVA to a lesser extend) and DNFB. The latter was
used to trigger inflammation in the mouse model and therefore suggests that inflammasome activation
in keratinocytes is likely to play a role in vivo as well[28].
In another study, inflammasome activation with DNFB has been shown to result in IL-1β release
and a decrease in expression of thymic stromal lymphopoietin (TSLP) in mouse and human
keratinocytes. TSLP skews an immune response toward a Th2 response which is associated with more
severe symptoms to allergens in atopic dermatitis and asthma[29]. The type(s) of inflammasome(s)
involved was not determined in this study. It should be noted that contrary to immune cells, pro-IL1β is constitutively expressed in keratinocytes[27]
A role for NLRP3 inflammasomes has also been suggested in atopic dermatitis. The potent dust
mite allergen Der p 1 has been shown in vitro to activate NLRP3 inflammasomes in keratinocytes. IL1β and IL-18 processing and release were increased by activation of the NLRP3 inflammasome by
Der p 1 whereas IL-1β and IL-18 expression levels remained unaffected. Inflammasome activation by
Der p 1 depends on its protease activity since it was unable to trigger inflammasome activation after
heat inactivation of the enzymatic activity. The inflammasome activation also required a K+ efflux as
high levels of extracellular K+ blocked inflammasome activation[30]. The mechanism by which Der
P 1 activates NLRP3 inflammasomes was not determined. A possible explanation could be that Der p
1 (partially) digests proteins on the surface of keratinocytes.
A role of inflammasomes in strong immune responses against foreign biological molecules is not
limited to the formation of NLRP3 inflammasomes in keratinocytes. The bee-venom component
melittin triggers AIM-2 but not NLRP3 inflammasome dependent IL-1β processing in human
keratinocytes. The activation of AIM-2 inflammasome was caused by a disruption of mitochondria by
melittin which results in leakage of mitochondrial DNA into the cytosol which is recognized by AIM2[31]. It is puzzling however why melittin did not disrupt the plasma membrane, which would result
in a K+ efflux and subsequent NLRP3 inflammasome activation. Additionally, Ca2+ release from
mitochondria has also been implicated in NLRP3 inflammasome activation[1].
Overall, these studies show that when keratinocytes are directly exposed to strong allergens they
can contribute to an inflammasome-dependent immune response. Both NLRP3 and AIM-2
inflammasomes have been shown to be functional in keratinocytes. However, the role of dedicated
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immune cells cannot be excluded based on these studies and I expect them to play a role as well.
Further study is required to determine their relative contribution to inflammasome-dependent immune
responses in different allergic reactions.
4. Response to endogenous proteins, metabolites and danger signals
Beside foreign activating signals, self-components such as proteins and metabolites can activate
inflammasomes in non-immune cells and this has been implicated to play a role in several disorders
including hyperhomocysteinemia, psoriasis, diabetes, age-related macular degeneration, ischemia/
reperfusion injury and atherosclerosis.
Hyperhomocysteinemia and psoriasis
Insufficient clearing of the metabolite homocysteine (a cysteine homologue), termed
hyperhomocysteinemia, can result in kidney inflammation and damage. It has been shown that
NLRP3 inflammasomes in podocytes become activated by homocysteine in a mouse model of
hyperhomocysteinemia. This results in the release of IL-1β and increased tissue damage [32]. The
inflammasome activation was shown to be dependent on NADPH for the generation of ROS[33].
However the dependence on NADPH for NLRP3 inflammasome activation has been challenged[1]. A
role for inflammasome activation in immune cells in response to hyperhomocysteine (in addition to
non-immune cells) cannot be excluded based on these results.
AIM-2 inflammasomes have been shown to play a role in the inflammatory skin disease psoriasis.
Increased IL-1β and activated caspase-1 were observed in skin lesions of psoriasis patients and AIM-2
protein expression levels were 50-fold increased as well. Microscopy imaging of skin tissue revealed
that the majority of keratinocytes in psoriatic lesions contain cytosolic DNA whereas this was not
observed for keratinocytes in healthy control skin[34].
Glucose and the NLRP3 inflammasome
The NLRP3 inflammasome is known to be activated in response to high levels of various metabolites
including glucose. This links the NLRP3 inflammasome to diabetes type II and associated pathology.
NLRP3 inflammasome activity has been shown in both immune and non-immune cells in response to
glucose. Stienstra et al. 2010 show that NLRP3 inflammasome dependent caspase-1 activity and
subsequent IL-1β and IL-18 release is increased in adipose tissue of obese mice compared to the
control and that this increase is likely derived from adipocytes rather than macrophages[35]. Caspase1 knockout mice have a lower body weight and smaller adipocytes compared to wildtype mice.
Additionally, adipose tissue from caspase-1 and NLRP3 knockout mice is more sensitive to insulin
and less sensitive to glucose compared to healthy control mice, suggesting a detrimental effect of
inflammasomes on diabetes[35]. A different study reports that knockout of the inflammasome
components NLRP3 and caspase-1 results in increased insulin sensitivity and glucose tolerance of
adipose tissue in obese mice compared to obese wildtype mice. However, contrary to Stienstra et al.
2010[35], this study suggest a primary role of NLRP3 inflammasome activation and IL1-1β
processing in macrophages rather than adipocytes in response to glucose[36]. It is interesting to note
that although both studies report a large decrease of IL-1β release in NLRP3[36] and caspase-1[35]
knockout mice IL-1β release is not completely ablated either. This suggests a role for canonicalinflammasome independent pro-IL-1β processing and possibly the involvement of inflammasomes
with other molecular sensors[37]. Mouse islets cells have also been shown to release IL-1β in
response to high levels of glucose in vitro. This release is largely dependent on NLRP3 and
thiorexodin-interacting protein (TXNIP). TXNIP is bound to thiorexodin in resting cells but
dissociates in response to high extracellular glucose levels and activates NLRP3. It should be noted
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that NLRP3, ASC and caspase 1 expression was much higher in bone-marrow derived macrophages
than in islet cells[38].
The NLRP3 inflammasome and age-related macular degeneration
Age-related macular degeneration (AMD) is a disease that can result in loss of central vision when it
progresses to more severe forms such as geographic atrophy (GA). It is characterized by the
accumulation of drusen. Drusen is an extracellular deposition of proteins (e.g. complement
components, amyloid-β), lipids and advanced glycation end products. In one study, six different
proteins present in drusen were tested for their ability to induce IL-1β release from the human retinal
pigment cell line ARPE-19. Only N-retinyliddene-N-retinylethanolamine (A2E) triggered IL-1β
release. Subsequent experiments show that this release is dependent on the NLRP3 inflammasome
and the enzymatic activity of the lysosomal protease cathepsin-B[39]. Surprisingly, amyloid-β (1-42)
did not trigger IL-1β release. Amyloid-β is known to result in lysosomal rupture and subsequent
NLRP3 inflammasome activation. It should be noted that the authors did not prime the cells to
upregulate IL-1β and inflammasome components and this may possibly explain why amyloid-β did
not activate inflammasomes in this study. A different study does show NLRP3 inflammasome
activation in response to the less toxic amyloid-β (1-40) in rat eyes. This activation was not
characterized by cell death in the used model[40].
Another characteristic of AMD is the accumulation of the noncoding retrotransposon RNA
sequence Alu. This accumulation of Alu RNA has been implicated in disease progression to GA by a
mechanism of NLRP3 inflammasome dependent cell death. Alu RNA results in ROS production and
subsequent NLRP3 inflammasome activation and cytotoxicity in retinal pigmented epithelium cells
(RPE) in both mice and humans. This pyroptosis-independent cytotoxicity is dependent on activation
of MyD88 and IL-18 but not on TLRs[41]. This is an interesting finding since this mechanism may
also exist in other celltypes. A more recent study shows that this NLRP3 activation is dependent on
the ATP-dependent ion channel P2X7[42]. The RNAse DICER1 was shown to have a protective role
against the Alu RNA induced cell death by cleaving Alu RNA. AMD is characterized by loss of
DICER1 expression. These findings were confirmed in vivo in RPE cell-specific DICER1 knockout
mice and by inhibition of inflammasome components. Additionally, increased caspase-1 activation,
released IL-18 and expression of NLRP3, ASC and caspase-1 were observed in the eyes of patients
with GA compared to eyes from healthy control[39]. Surprisingly, the observed cell death was
independent of pyroptosis. Also RIG-1 activation via a different double stranded RNA species did not
result in cell death. I would expect RIG-I inflammasome formation to also result in caspase-1
activation and IL-18 processing. This suggests NLRP3 inflammasome formation activates additional
signals that are required for MyD88 and IL-18 dependent cell death as well. Alternatively, additional
exogenous signals may be required for RIG-I and/or AIM-2 inflammasome activation and subsequent
cell death.
A different study reports a protective effect of IL-18 on the development of wet AMD in a mouse
model of laser induced choroidal neovascularization (CNV). IL-18 and NLRP3 knockout mice have
increased lesions volume compared to wt mice. The amount of IL-1β released by ARPE-19 cells was
also compared to the amount of IL-1β released by PBMCs in response to ATP after LPS priming.
PMBCs released ~25 fold higher levels ofIL-1β compared to ARPE-19 cells and the authors conclude
from this that infiltrated myeloid cells are likely to be mainly responsible for IL-1β and IL-18 release
in the retina[43].
Collectively, these studies strongly suggest the involvement of NLRP3 inflammasomes in nonimmune cells during AMD. Whether this has a detrimental or protective effect on disease progression
is less clear. Doyle et al. 2012. Observed a protective effect of IL-18[43] whereas Tarallo et al. 2012
observe a detrimental effect on AMD progression[41]. Both studies use a different model and readout
10
for the effect on disease, which may help to explain the observed differences. Whether IL-18 is
detrimental or protective for disease progression could also be dependent on the stage of the AMD
progression. More studies are required to determine the effects of the observed inflammasome
activation and to better characterize the relative role of non-immune cells in AMD compared to
immune cells.
Ischemia and reperfusion injury
A role for inflammasomes has also been implicated in ischemia/reperfusion (I/R) injury. Kawaguchi
et al. observed less I/R injury in ASC knockout mice compared to wildtype mice after a 30 minute
occlusion of the left anterior descending artery (a branch of the coronary artery) followed by 48 hours
of reperfusion[37]. Heart function was also improved and less scarring was observed in ASC
knockout mice compared to wt mice at 7 and 14 days after induced myocardial infarction. Similar
results were observed with caspase-1 knockout mice. Adoptive bone marrow experiments with ASC
knockout mice show that inflammasomes in both immune and non-immune cells are involved in I/R
injury. In vitro experiments indicate that cardiac fibroblasts but not cardiomyocytes display
inflammasome activation during I/R injury. IL-1β release from cardiac fibroblast but not from
cardiomyocytes could be induced with various stimuli (hypoxia/reoxygenation, the K+ ionophore
nigericin)[37]. The inflammasome type was not experimentally confirmed, but considering the stimuli
they used, the inhibition of IL-1β release by extracellular K+ and the observed production of ROS
suggest that the NLRP3 inflammasome is likely to be involved. A more recent study confirms that the
NLRP3 inflammasome is involved here. Additionally, they exclude a role in inflammasome activation
for infiltrated immune cells in the cardiac tissue by using an ex vivo langendorf model [44].
Contrary to Kawaguchi et al.[37], Mezzaroma et al. did observe (NLRP3) inflammasome
activation in the mouse cardiomyocyte cell line HL-1 in response to ATP with LPS priming and
hypoxia[45]. This also resulted in cell death dependent on caspase-1 activity since an inhibitor of
caspase-1 reduced cell death[45]. This cell death is likely to be pyroptosis. The seemingly conflicting
results on the role of cardiac myocytes may be explained by the use of a different concentration of
ATP. Kawaguchi et al. use an ATP concentration of 10uM[37] whereas Mezzaroma et al. use
between 1 and 5 mM[45]. The stimuli that did trigger inflammasome activation in cardiac fibroblasts
but not in cardiomyocytes were not tested by Mezzaroma et al. Sandanger et al. also observe
inflammasome activation in primary mouse cardiac fibroblasts in response to ATP at a concentration
of 3mM[44] further indicating the concentration may have been too low. Alternatively the observed
differences may be caused by characteristics of the HL-1 cell line and primary cardiomyocytes.
Atherosclerosis
One known promoter of atherosclerosis development is oscillatory shear flow. It has been shown in
vitro that oscillatory shear flow upregulates the expression of Sterol regulatory element-binding
protein 2 (SREBP2) which indirectly activates the NLRP3 inflammasome. SREBP2 expression results
in increased NADPH oxidase expression, which in turn results in ROS production which triggers
subsequent NLRP3 inflammasome-dependent IL-1β processing. This study also suggests this occurs
in vivo, since they observed increased activated caspase-1 and processed IL-1β in the aortic arch in
mice (where higher oscillatory shear flow occurs due to the bend and branches) compared to the
thoracic aorta[46].
This observed role of SREBP seems to contradict a previous study where SREBP1 and 2were
found to be activated by inflammasome activation resulting in membrane biogenesis[16]. This could
indicate a celltype specific role for SREBP2. Alternatively, if both functions apply to the same
celltypes, it could be a positive feedback loop. A different study observes increased IL-1β release and
caspase-1 activity in vitro in primary rat vascular smooth muscle cells that were calcified with β11
glycerophosphate. This increase is dependent on the NLRP3 inflammasome since knockdown of
NLRP3 reduced IL-1β release and caspase-1 activation. They also observed increased caspase-1
activity in calcified human arteries compared to non-calcified arteries in the same patient, suggesting
that calcification of arteries may also activate inflammasomes in humans[47]. These studies suggest
that the NLRP3 inflammasome is activated by oscillatory shear flow and vessel calcification.
All of the studies on inflammasome activation in non-immune cells in response to self-molecules
found a primary role for NLRP3 inflammasomes except in the case of psoriasis where AIM-2 was
identified as the molecular sensor. Additionally, all of the activating signals appear to be danger
signals. Inflammasome activation appears to result in more severe disease progression in diabetes,
hyperhomocysteinemia, ischemia/reperfusion injury and possibly atherosclerosis. Most studies on
inflammasomes in AMD also suggest detrimental effect of inflammasome activation on AMD
progression. However, Doyle et al. 2012 report a protective effect[43].
5. Response to non-biological substances and stimuli
Inflammasome activation in non-immune cells also occurs in response to wide range of substances,
such as asbestos, urban particle matter, cigarette smoke and titanium dioxide. Many of these
substances can be encountered in everyday life. Beside chemical substances, exposure to UVB also
results in inflammasome activation in non-immune cells.
Urban particle matter results in NLRP3 and caspase-1 dependent release of IL-1β in human airway
epithelial cells[48]. Exposure of wildtype mice to urban particle matter also resulted in increased
levels of IL-1β in bronchoalveolar lavage fluid compared to unexposed mice. This effect was not
observed in NLRP3 knockout mice. Whether the observed increase in IL-1β is caused by immune
cells, pulmonary epithelial cells or both has not been determined. The mice displayed only very few
clinical symptoms of pulmonary inflammation[48]. Another study shows NLRP3 dependent
inflammasome activation and IL-1β release in a human bronchial epithelial cell line (BEAS-2B) and
primary human bronchial epithelial cells in response to silica particles. A much higher (22 fold) IL-1β
release was observed in THP-1 cells compared to BEAS-2B cells in response to the same
concentration of silica particles[49].
Titanium dioxide nanoparticles, which are frequently used as a white pigment, also trigger
inflammasome activation. However, the role of non-immune cells is not fully characterized. Exposure
of cultured human keratinocytes and leukemic monocyte THP-1 cells to titanium dioxide
nanoparticles resulted caspase-1 activation and IL-1β processing. This response was shown to be
dependent on NLRP3 in mouse and human bone marrow-derived macrophages. However, prolonged
exposure of mouse skin to titanium dioxide nanoparticles did not result in clinical signs of
inflammation. They did observe epidermal thickening which may indicate increased proliferation.
Pulmonary exposure did result in inflammation, but whether non-immune cells also play a role here
was not determined[50].
Cigarette smoke has been shown to trigger IL-1β release in a cultured human bronchial epithelial
cell line (HBE-14o). This release was blocked by a caspase-1or a P2x7 inhibitor and also by a ROS
scavenger. It was not determined whether ASC and which molecular sensor(s) are involved.
Considering that inhibition of P2x7 or a ROS scavenger inhibited IL-1β release, it appears likely that
the NLRP3 inflammasome is involved[51].
Cultured human mesothelial cells exposed to crocidolite (a type of asbestos) or the mineral
erionite, which has similar properties to asbestos in some regards, show increased caspase-1 activity
and IL-1β and IL-18 release compared to control cells. This increase was inhibited by NLRP3
knockdown. Treatment with the IL-1 receptor antagonist Anakinra reduced IL-1β release, suggesting
autocrine stimulation[52].
12
Non chemical stimulation in the form of UVB radiation has also been reported to result in
inflammasome activation in keratinocytes. Stimulation of primary human keratinocytes with UVB
results in IL-1β release but not the release of IL-18. The IL-1β release was inhibited by the
knockdown of caspase-1, ASC, NLRP1 or NLRP3 showing the involvement of the NLRP1 and
NLRP3 inflammasome. The IL-1β release was also inhibited by a calcium chelator, indicating the
dependence on a calcium influx. Additionally, a reduced number of neutrophils were detected in UVB
irradiated skin of caspase-1 knockout mice compared to wildtype mice, suggesting a role for
inflammasomes in recruiting them to the site of inflammation[53]. A more recent study also
determined that UVB-induced IL-1β release from keratinocytes is dependent on activation of human
caspase-4 but not caspase-5. Caspase-4 and -5 are likely to be orthologs of mouse caspase-11[54].
A number of studies report on NLRP3 inflammasome activation in lung epithelial cells and on
NLRP1 and NLRP3 inflammasome activation in keratinocytes. However, whether inflammasome
activation in response to these stimuli has beneficial or pathological consequences is not well
characterized. In vivo studies will be required to determine this. It should also be noted that
keratinocytes in the skin are normally not directly in contact with most external stimuli, since they are
covered by layers of dead keratinocytes. This may also explain why exposure of mouse skin to
titanium dioxide nanoparticles did not result in inflammation whereas pulmonary exposure in mice
did[50].It is interesting to note that all of the above mentioned stimuli are known to or have been
implicated to be carcinogenic. Since inflammasomes are known to be involved in carcinogenesis (see
below) there could be a mechanistic link here.
6. Inflammasomes in wound healing, tumor development and pain sensitivity
Effects of inflammasome activation have also been reported in the development in tumors and cell
proliferation in skin and the intestine. Most of these studies report a protective effect of
inflammasomes against tumor formation. However, some studies report that inflammasomes may
promote tumor development. One study shows an inflammasome-independent role for ASC in
keratinocytes in suppressing tumor formation. A number of studies also suggest a role for
inflammasome activation in promoting or repressing cell proliferation and wound healing.
Wound healing and tumors
Caspase-1 knockout mice have an increased number and severity of tumors in the colon in a model of
chemically induced tumorigenesis compared to wt mice. Increased proliferation of colon epithelial
cells and reduced cell death in tumors of caspase-1 knockout mice was observed compared to
wildtype control mice. The increased number of tumors and tumor severity was observed in NLRC4
knockout mice but not NLRP3 mice. This suggests a role for NLRC4 but not NLRP3 in this
model[55]. The underlying activating signal for NLRC4 inflammasome activation has not been
elucidated. The authors hypothesize based on the well-established activation of NLRC4 by Gramnegative bacteria that altered microbiota in the colon may play a role. Although the increased
proliferation was observed in colon epithelial cells, a role for immune cells in the observed knockout
phenotypes cannot be excluded. The observed decrease in cell death in tumor cells may possibly be
caused by deficient pyroptosis.
In another study, reduced colon healing was observed in NLRP6 knockout mice after causing
biopsy injury. There was also an increase in the number of tumors in NLRP6, ASC and caspase-1
knockout mice in a model of chemically induced tumorigenesis. Adoptive transfer of bone-marrow of
caspase-1 knockout or wildtype into caspase-1 knockout or wildtype mice reveal that caspase-1 is
required in both hematopoietic and non-hematopoietic cells to acquire the tumor phenotype of
caspase-1 knockout mice, since neither of the partial knockouts was sufficient to acquire the
13
phenotype[56]. This study suggests that inflammasome activation in non-immune cells together with
inflammasomes activation in immune cells can promote tumorigenesis and wound healing. A
mechanistic link between inflammasome activation and the observed phenotypes has not been found
yet.
Another study reports a protective effect of inflammasome activation on tumorigenesis but
concludes this is primary caused by inflammasome activation in immune cells. Experiments with
adoptive transfer of bone marrow of NLRP3 knockout and wildtype mice show that NLRP3
inflammasome activation in hematopoietic cells is primarily responsible for the observed protective
effect of the inflammasome on tumorigenesis[57]. These results do not exclude the possibility that
another molecular sensor may be activated in non-hematopoietic cells in this model. In a different
study, IL-18 has been shown to promote proliferation of intestinal epithelial cells in mice by indirectly
increasing IL-22 expression[58]. IL-18 downregulates the expression of IL-22bp. IL-22bp is
expressed mostly in hematopoietic cells and inhibits IL-22. IL-22 promotes proliferation of intestinal
epithelial and tumor cells. They also show that the expression of IL-22bp is dependent on NLRP3 and
NLRP6 but not NLRC4. Therefore, inflammasome activation results in IL-18 release, which reduces
IL-22bp expression in hematopoietic cells, which in turn abolishes inhibition of IL-22 which
subsequently results in increased proliferation of intestinal epithelial and tumor cells[58]. This study
provides a mechanism on how inflammasome activation may promote tumorigenesis and wound
healing in the intestine. This study also reports that the microbiota affects the downregulation of IL22bp[58]. Whether the inflammasome activation occurs in epithelial or hematopoietic cells is not
known in this study. A role for inflammasomes in promoting wound healing in the skin has also been
implicated by affecting the release of processed IL-1α[12].
One study shows an inflammasome-independent role for ASC as a tumor suppressor in
keratinocytes. At the same time they show an inflammasome-dependent tumor promoting role in
immune cells. Reduced skin tumor formation after treatment with carcinogenic substances in IL-1R
and caspase-1 knockout mice was observed compared to the wt control. However, this was not
observed for ASC knockout mice. They then used myeloid and keratinocyte specific ASC knockout
mice to try to explain these seemingly conflicting results. Myeloid ASC knockout mice show fewer
tumors whereas keratinocyte ASC knockout mice have more tumors compared to wildtype control
mice. A reduction in processed IL-1β was observed in tumors of myeloid ASC knockout mice
compared to the control whereas no difference between the wt control was observed for keratinocyte
ASC knockout mice tumors. This suggests that infiltrating myeloid cells are the primary source of
processed IL-1β rather than keratinocytes in this model of skin tumors. In vitro experiments revealed
increased proliferation in keratinocytes from ASC knockout mice compared to the control whereas
caspase-1 and NLRP3 knockout keratinocytes did not. They also observe decreased ASC expression
in tumors compared to healthy skin. Additionally, they observe reduced P53 phosphorylation and
downstream p21 and Notch1 expression in keratinocytes from knockout mice compared to
keratinocytes from healthy control mice. Together, these data suggest an inflammasome independent
role for ASC as a tumor suppressor in keratinocytes which may be mediated by regulating P53
activity[6]. Inflammasomes do not appear to play a role in keratinocytes in this study despite the many
studies that do observe inflammasome activation and IL-1β processing in keratinocytes. Infiltrating
myeloid cells on the other hand do show inflammasome-dependent IL-1β release in these tumors. This
observed cell type specific inflammasome activation could be mediated by additional control factors
that are differentially expressed in different cell types.
Pain sensitivity
Inflammasome dependent processing of IL-1β and IL-18 in keratinocytes has also been implicated in
increasing pain sensitivity. In a rat model of tibia fracture, increased NLRP1 and caspase-1 expression
14
was observed in keratinocytes in the skin around the fracture and also elevated levels of released IL1β and IL-18 compared to non-fractured limbs. Injection of IL-18 and IL-1β in a limb also increased
the sensitivity to mechanical pain induction. The inhibition of caspase-1 decreases the enhanced pain
sensation in a limb that has previously been fractured compared to a non-fractured limb[59]. They did
not determine the activating signal for inflammasomes or confirm that NLRP1 inflammasomes
process IL-1β and IL-18.
Taken together, these studies show that inflammasomes in non-immune cells as well as in immune
cells are likely to be involved in tumor formation and wound healing in the skin and the intestine.
Beside these processes, inflammasome activation in keratinocytes has also been implicated in
increasing pain sensitivity. Whether inflammasome activation stimulates or diminishes wound healing
and tumor formation is less clear. Most studies report a stimulatory effect of inflammasomes on
proliferation. However, Hu et al. 2010 report a decline in proliferation of colon epithelium[55]. Most
studies show a decline in tumor formation when inflammasomes are still functional. However, Huber
et al. 2012 report a stimulating effect of inflammasomes in either immune and/or non-immune cells
on tumorigenesis[58] and Drexler et al. 2012 also report a stimulating effect of inflammasomes in
immune cells on tumorigenesis (but an inflammasome-independent protective role for ASC against
tumor formation in non-immune cells)[6]. All the studies that report a protective effect of
inflammasome activation on tumor formation conclude that this is caused by inflammasomes in nonimmune cells. This could suggest that inflammasome activation in non-immune cells has a protective
effect against tumorigenesis whereas inflammasome activation in immune cells promotes tumor
formation. Additional regulatory mechanisms may also be involved which may help explain the
different results in these studies. Inflammasome activation may also have a protective or stimulating
effect on tumors depending on the stage of tumor development. A role for inflammasomes in affecting
microbiota, which in turn may affect tumor development has also been suggested but no yet
investigated.
15
Overview of inflammasome types in non-immune cells
Cell type(s)
species
In vivo or
in vitro
Inflammasome
types
Activating signals
Possible effect of inflammasome
activation
references
mouse
In vivo
NLRP3
Microbiota
Changes in microbiota, less sever NASH
symptoms
[21]
Mouse
in vivo
NLP6
Microbiota
Less severe colitis
[22]
Colon epithelial cells
Mouse
In vivo
NLRC4
reduced proliferation
[55]
Colon epithelial cells
Mouse
In vivo
NLRP6
increased proliferation
[56]
intestinal epithelial and goblet cells
Mouse
In vivo
NLRP6
Increased mucus secretion
[26]
Caco-2
Human
In vitro
NLRP3
Y. enterocolitica
IL-18 release (but no IL-1β)
[23]
Human
In vitro
NLRP3
Chlamydia trachomatis
Increased c. trachomatitis infectivity in vitro
[17]
Primary lung epithelial cells
human
In vitro
NLP3, RIG-I
Influenza A
Lung epithelial cells
Mouse
In vivo
NLRP3
OVA
Inflammation
[60]
HBE-14o
Human
In vitro
NLRP3 (?)
Cigarette smoke
Inflammation
[51]
Airway epithelial cells
Human
In vitro
NLRP3
Urban particle matter
Inflammation
[48]
BEAS-2B and primary human bronchial
epithelial cells
Human
In vitro
NLRP3
Silica particles
Suggestive for effect in silicosis
[49]
Mesothelialial epithelium cells
Human
In vitro
NLRP3
Asbestos, erionite
inflammation
[52]
Adipocytes
Mouse
In vivo
NLRP3
sucrose
increased glucose sensitivity, decreased insulin
sensitivity, possible role diabetes
[35]
Primary adipocytes
Human
In vitro
Sucrose
Possible role diabetes
[61]
Keratinocytes
Mouse
In vitro
NLRP3 (?)
UVB, SDS, DNFB
Possibly increased inflammation early phase of
contact hypersensitivity
[28]
Primary human keratinocytes
Human
In vitro
NLRP3
Der p 1
Possible role in atopic dermatitis
[30]
Intestinal tract
Non-hepatic non-hematopoietic cells(presumably
intestinal epithelial cells)
Non-hematopoietic cells (presumably intestinal
epithelial cells)
Cervix
HeLa cells
Lung
[4]
Adipose tissue
Skin
16
Primary human and mouse keratinocytes
Human
and mouse
In vitro
?
DNFB
Keratinocytes
Human
In vitro
AIM-2 (?)
Double stranded DNA
Melittin, mitochondrial
and nuclear DNA
DNA localized in
cytosol
Possible stimulatory role in atopic dermatitis
and increased Th2 skewing
[29]
[62]
Inflammation bee sting, possible role
hypersensitivity
[31]
Possible role in psoriasis
[34]
Increased pain sensitivity
[59]
Keratinocytes
Human
In vitro
AIM-2
Keratinocytes
Human
In vivo
AIM-2
Keratinocytes
Rat
In vivo
NLRP1 (?)
Keratinocytes
Human
In vitro
NLRP3 (?)
Titanium dioxide
nanoparticles
inflammation
[50]
Keratinocytes
Human
In vitro
NLRP3, NLRP1
UVB
Neutrophil influx to site of inflammation
[53]
Mouse
In vivo
NLRP3
Homocysteine
Hyperhomocysteinemia progression
[32]
ARPE-19
Human
In vitro
NLRP3
A2E, ATP
AMD progression
[39]
RPE cells
Human
and
Mouse
In vivo
NLRP3
Alu RNA
AMD progression
[41][42]
ARPE-19
Human
In vitro
NLRP3
ATP
Protects against AMD
[43]
cardiac fibroblasts
Mouse
In vivo and
in vitro
? (NLRP3?)
Nigericin, hypoxia
Ischemia/reperfusion injury
[37]
HL-1
Mouse
In vitro
NLRP3
ATP
Cell death
[45]
Cardiac myocytes
Mouse
NLRP3
ATP
Ischemia/reperfusion injury
[44]
Endothelial cells
Human
and mouse
In vitro and
ex vivo
In vivo and
in vitro
NLRP3
Oscillatory shear flow
Possible role in atherosclerosis
[46]
Vascular smooth muscle cells
Rat
in vitro
NLRP3
Calcium phosphate
deposits
Possible role in atherosclerosis
[47]
endothelial telomerase-immortalized human
umbilical cells
Human
In vitro
IFI16
KSHV genome
Inflammation induced by pathogen
[10]
Islet cells
Mouse
In vitro
NLRP3
Sucrose
Possible role in diabetes
[38]
Kidney
Podocytes
Eye
Heart and blood vessels
Other
Table 1. Overview of inflammasome types, stimulatory signals and effects of inflammasome activation in non-immune cells. A question mark indicates that the
inflammasome type has not been fully confirmed. The in vitro/in vivo indicates whether the inflammasomes in non-immune cells were specifically identified in non-immune
cells in vivo or in vitro.
17
7. Discussion
Ample evidence exists for a role of inflammasomes in non-immune cells. Inflammasome activation in
non-immune cells have been implicated as a response against various stimuli including pathogens, the
microbiota, metabolites, self-proteins and self RNA species, hyper immune responses against foreign
substances (biological and non-biological), wound healing and cancer. Different molecular sensors of
inflammasomes have been identified in non-immune cells including NLRP1, NLRP3, NLRC4,
NLRP6, AIM-2, RIG-I and IFI16. NLRP3 is the most frequently involved in these studies.
Most of the studies discussed in this review focus on canonical inflammasomes and interleukin
production. Caspase-11-dependent inflammasome activation, caspase-8 dependent IL-1β processing,
the role of IL-1α as an IL-1R ligand and alternative inflammasome functions are often not taken into
consideration. Besides upregulation of inflammasome components (priming), additional regulatory
mechanisms of inflammasome activation are often ignored as well. Many studies have inconsistent
results regarding the in vivo effect of inflammasome activation on disease. Investigation of additional
mechanisms involved in inflammasome activation may help to explain these discrepancies. The
inconsistent results may also indicate that inflammasome activation can be beneficial or detrimental
depending on the situation or that the right balance in inflammasome activation is required.
The same types of inflammasomes are present in immune and non-immune cells. Inflammasome
activation in non-immune cells also results in the processing of pro-interleukins as observed in
immune cells. Studies that compare the amount of IL-1β released by immune cells to non-immune
cells for the same number of cells in vitro[24][49][43] or for the total contribution in an organ or
tissue in vivo[25][6] conclude that immune cells release more processed IL-1β. Since many epithelial
cells can be continuously exposed to inflammasome stimulating substances, a lower sensitivity to
these stimuli may prevent continues inflammasome activation. Despite the lower pro-interleukin
processing of non-immune cells, many studies see potent effects of inflammasome activation in nonimmune cells on disease. This suggests that alternative functions of inflammasome activation may
mediate these effects. Another possibility is that inflammasome activation in non-immune cells and
IL-1β release result in recruitment of immune cells which then further amplify inflammation. Many
epithelial tissues have been shown to express functional inflammasomes. Epithelial cells are likely to
be the first cells exposed to pathogens and foreign molecules and are therefore likely candidates to
initiate inflammation. One study also observed a reduced influx of neutrophils to UVB irradiated skin
area in keratinocyte specific caspase-1 knockout mice compared to wildtype mice[53], which supports
this theory. Experiments with cultured mesothelial epithelium cells also suggest a role for IL-1β in
autocrine signaling to further amplify inflammation[52].
A large number of exogenous stimuli that are frequently encountered in daily life as well selfproteins and metabolites have been shown to be able to trigger inflammasomes in non-immune cells.
Many of these signals could be characterized as danger signals. If the primary function of
inflammasome activation is to detect pathogens and to initiate an immune response, it seems
unbeneficial to do so without detecting a pathogen-specific signal as well when considering the
potential damage of inflammation. One explanation could be that it’s an evolutionary tradeoff where
the risk of not detecting a pathogen early is greater than the damage caused by occasional accidental
inflammasome activation. Alternatively, it could indicate that many of these stimuli do not
accidentally activate inflammasomes but instead function to activate alternative functions of
inflammasomes, such as proliferation or pyroptosis. All of the non-biological exogenous stimuli
discussed in this review are known or very likely to be carcinogenic. Since inflammasome activation
has been implicated in increased pain sensitivity[59], inflammasome activation could be a mechanism
to cause irritation to trigger a behavioral change to avoid exposure to dangerous substances.
Inflammasome activation in response to substances that can cause direct tissue damage may be
18
beneficial to initiate tissue repair and prepare the immune systems for any pathogens that may now be
able to pass over the epithelial barrier.
Effects of inflammasome activation in non-immune cells on many diseases have frequently been
observed. This shows that inflammasome activation in non-immune cells has biological relevance in
vivo. Many studies report conflicting results on whether inflammasome activation is beneficial or
detrimental in diseases. This indicates a more complex situation then our current understanding. More
research is required to determine the functions and effects of inflammasome activation in non-immune
cells as well as to better understand non-canonical inflammasomes. Also the relative role of
inflammasome activation in non-immune cells compared to immune cells as well as their interplay is
currently still poorly understood. A singular role for inflammasomes in immunity does not seem likely
in the light of these studies.
References
[1] E. Latz, T. S. Xiao, and A. Stutz, “Activation and regulation of the inflammasomes.,” Nature reviews.
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