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Activation of the NLRP3 inflammasome by particles from the 1
Echinococcus granulosus laminated layer 2
Cecilia Casaravilla1, Álvaro Pittini
1, Dominik Rückerl
2, Judith E. Allen
2 3
& Álvaro Díaz1*
4
1. Área Inmunología, Departamento de Biociencias (Facultad de Química) and Cátedra 5
de Inmunología, Instituto de Química Biológica (Facultad de Ciencias), Universidad de 6
la República, Montevideo, Uruguay. 7
2. Faculty of Biology, Medicine and Health, School of Biological Sciences, University 8
of Manchester, M139PT, Manchester, UK. 9
10
(*) Corresponding author. E-mail: [email protected]. Postal address: Dr. Álvaro Díaz. 11
Cátedra de Inmunología. Instituto de Higiene. Avenida Alfredo Navarro 3051. 12
Montevideo CP11600. Uruguay. Tel: + 59824874320. 13
14
Running title: NLRP3 inflammasome activation by helminth material 15
Keywords: Echinococcus; laminated layer; alum; NLRP3; PI3K; membrane affinity-16
triggered signaling 17
18
IAI Accepted Manuscript Posted Online 22 June 2020Infect. Immun. doi:10.1128/IAI.00190-20Copyright © 2020 Casaravilla et al.This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license.
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ABSTRACT 19
The interaction of dendritic cells and macrophages with a variety of rigid non-cellular 20
particles triggers activation of the NLRP3 inflammasome and consequent secretion of 21
IL-1β. Non-cellular particles can also be generated in the context of helminth infection, 22
as these large pathogens often shed their outermost structures during growth and/or 23
moulting. One such structure is the massive, mucin-based, soft and flexible laminated 24
layer (LL), which protects the larval stages of cestodes of the genus Echinococcus. We 25
show that particles from the E. granulosus LL (pLL) trigger NLRP3- and caspase-1-26
dependent IL-1β in LPS-primed mouse bone marrow-derived dendritic cells (BMDC). 27
This response can be elicited by pLL particles too large for phagocytosis, and 28
nonetheless requires actin dynamics, Syk and PI3K. These three requirements had 29
already been observed in our previous study on the alteration by pLL of BMDC 30
responses to LPS in terms of CD86, CD40, IL-10 and IL-12: however, we now show 31
that these alterations are independent of NLRP3 and caspase-1. In other words, an initial 32
interaction with particles requiring actin dynamics, Syk and PI3K but not phagocytosis 33
elicits both NLRP3-dependent and NLRP3-independent responses. Intraperitoneal 34
injection of pLL induced IL-1, suggesting that contact with LL materials induces IL-35
1 in the E. granulosus infection setting. Our results extend NLRP3 inflammasome 36
activation by non-cellular particulate materials both to helminth-derived and to 37
flexible/soft materials. 38
39
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INTRODUCTION 40
The NLRP3 inflammasome is an innate immune sensor-response modulus triggered by 41
an exceptionally wide range of stimuli (1, 2). Recent works suggest that it plays 42
important roles in helminth infections, antagonizing type 2 responses and potentiating 43
Th17/inflammatory responses, with impacts on parasite burden and pathology (3-8). In 44
addition and counter-intuitively, NLRP3 can promote type 2 responses, independently 45
of the inflammasome (9, 10). 46
The possible triggers for NLRP3 inflammasome activation in helminth infections 47
include endogenous signals generated by inflammation and tissue damage and helminth 48
products themselves (8). Helminth products shown to trigger the NLRP3 inflammasome 49
so far are all either soluble or exosomal (3, 4, 7, 11). Outside of the field of helminth 50
infection, rigid particulate matter, specifically of crystalline nature, is well known to 51
activate the NLRP3 inflammasome in macrophages and DCs previously stimulated 52
(primed) with TLR agonists (1, 2, 12-24). Helminths, as a result of the turnover of their 53
outermost structures, have much potential to generate insoluble matter within host 54
tissues. However, such matter is physically very different from the crystalline materials 55
known to activate the NLRP3 inflammasome; in fact, the possibility of NLRP3 56
inflammasome activation by insoluble helminth materials has not been analyzed. One 57
such outermost helminth structure is the laminated layer (LL), a mm-thick mucin-based 58
protective structure of the Echinococcus granulosus sensu lato (s.l.) larva (25-27). This 59
bladder-like larva causes cystic echinococcosis in livestock and humans (28-30). Larval 60
growth is accompanied by shedding of LL particles, observed in E. granulosus s.l. 61
experimental infections (31), and better documented for the closely related species E. 62
multilocularis (32-34). 63
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We have previously analyzed the immunological effects of a preparation of particles 64
from the E. granulosus s.l. LL (termed pLL) as a possible model of LL particles shed in 65
vivo (35-37). pLL particles are made up of an aqueous gel, and are soft and deformable 66
(35). In mouse bone marrow-derived dendritic cells (BMDC) in particular, pLL induces 67
the expression of CD86, and enhances LPS-elicited CD86 and IL-10 while blunting the 68
response to LPS in terms of CD40 and IL-12p70 (as well as its subunit IL-12/23p40) 69
(35). These changes elicited by pLL require actin dynamics, PI3K class I and probably 70
the kinase Syk but not particle phagocytosis, and appear to be receptor-independent 71
(36). These features match a mechanism termed “membrane affinity-triggered 72
signaling” (MATS), put forward by Yan Shi to explain DC and macrophage responses 73
to solid, mostly crystalline materials (38). In this proposed mechanism, solid surfaces 74
interact with polar headgroups of certain plasma membrane lipids causing the 75
coalescence of lipid rafts and/or specifically the aggregation of phosphatidylinositol 4,5-76
bisphosphate (PIP2) (38-41). The cytosolic protein moesin is then recruited to clustered 77
PIP2 and in turn causes activation of Syk and downstream signaling that does not 78
require conventional receptors (41). MATS signaling may trigger phagocytosis, but it 79
can operate from the cell surface in the absence of particle internalization (39-41). 80
Materials proposed to act on DCs via MATS include sodium urate and alum (39-41), 81
which are additionally known to activate the NLRP3 inflammasome (13, 15). 82
The mechanistic similarities between responses to pLL and those induced by MATS led 83
us to hypothesize that pLL could also activate the NLPR3 inflammasome; we also 84
wondered if such activation may underlie the changes caused by pLL on BMDC 85
responses to LPS (35, 36). In this paper we show that pLL does elicit NLRP3-dependent 86
IL-1β from BMDC, but the previously described alterations in BMDC responses to LPS 87
are NLRP3-independent. We also show that NLRP3 inflammasome activation by pLL 88
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shares the MATS-like requirements with the previously described alterations to LPS 89
responses, adding weight to the hypothesis that DC recognition of pLL involves a 90
MATS-like interaction. Our results extend the range of particulate NLRP3 91
inflammasome activators to soft/flexible materials, and suggest that additional insoluble 92
materials shed by helminths may activate the hosts’ NLRP3 inflammasome module. 93
94
Materials and Methods 95
Parasite materials. pLL, pLL treated for reduction/alkylation of disulphides, and non-96
phagocytosable pLL (pLLNP
) were generated, had their concentrations determined, and 97
were stored as described (35, 36). Preparation of pLL involves a dehydration step 98
followed by grinding into a fine powder, re-hydration and filtration of the resulting 99
suspension; of the two dehydration methods described in (35), freeze-drying was used 100
in the present work. pLL preparations tested negative for endotoxin by the Limulus 101
amebocyte lysate (LAL) method (35). 102
BMDC generation and stimulation. GMCSF-BMDC were generated as described (35, 103
36), from female C57BL/6 wild-type or NLRP3 gene-deficient mice (B6.129S6-104
Nlrp3tm1Bhk
/J; Jackson Laboratories). For inflammasome activation assays, BMDC 105
(400,000 cells per well of 96-well plates) were primed for 2 h with 10 ng/mL LPS (from 106
E. coli 0127:B8; Sigma) or incubated with medium only (final volume in priming step: 107
100 µL). Then cells were stimulated with pLL, pLLNP
, alum (AllhydrogelTM
, Invivogen) 108
or ATP (Sigma), or medium only (100 µL additional volume). Supernatants were 109
collected 3 h later. For assays determining effects on LPS-induced BMDC 110
costimulatory molecule expression and cytokine output, pLL or alum was added (final 111
volume in this step was 100 µl) followed 1 h later by LPS (10 ng/mL, in 100 µL 112
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additional volume) and cells and supernatants were harvested 18 h later (35). Cell 113
viability was measured on the basis of exclusion of the ToPro3 (Invitrogen; 50 nM)
114
viability probe in flow cytometry. 115
Generation and stimulation of bone marrow-derived macrophages (BMDM). BMDM 116
were generated from the bone marrow of female C57BL/6 mice by differentiation 117
during 7 days in the presence of L929 cell supernatant as a source of M-CSF, as 118
described in (42). Cells were stimulated for inflammasome activation as described for 119
BMDC, except that they were plated at 200,000 cells per well of 96-well plates, and the 120
doses of pLL and alum used were at 50 and 50 µg per million cells respectively. 121
Chemical inhibitors. The following chemical inhibitors were used: Z-YVAD-FMK (10 122
µM; Calbiochem/Merck), wortmannin (Sigma; 1 µM) GDC-0941 (5 µM; 123
Calbiochem/Thermo), piceatannol (25 µM; Santa Cruz Biotechnology), cytochalasin D 124
(5 µM; Sigma-Aldrich), VPS34-IN1 (1 µM) and SAR405 (1 µM) (both from the 125
Division of Signal Transduction Therapy Unit, University of Dundee). 126
Measurement of cell responses. Cytokines in supernatants were measured with 127
commercial ELISA kits from RnD Systems (DuoSet ELISA kit for IL-1β and IL-10), 128
eBioscience (IL-18 mouse ELISA kit), or with an antibody pair formed by unconjugated 129
antibody from BD Pharmingen and a biotinylated antibody from Biolegend (IL-130
12/23p40). The expression of CD40 and CD86 was measured by flow cytometry in cells 131
gated for CD11c expression, as in (35). 132
Measurement of Akt phosphorylation. BMDC were stimulated with LPS (10 ng/mL) 133
alone or together with pLL or alum during 80 min (36). Phosphorylation of Akt at Ser473
134
was measured by Western blot as described (36, 37). 135
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In vivo effects of pLL. C57BL/6 mice (female, 8-10 weeks old) were injected i.p. with 136
pLL (150 µg dry mass per mouse), LPS (15 µg per mouse), both pLL and LPS, or 137
vehicle only (200 µl of PBS). Three h later, mice were euthanized using isofluorane and 138
peritoneal lavage fluid was collected for IL-1β quantification. 139
Statistics. 140
Data were analyzed by a non-parametric method, thus avoiding having to assume 141
normality and homogeneity of variances. Specifically, the extension of the Friedman 142
test with the Conover post-test and the Bonferroni correction was applied (43). This 143
method incorporates data corresponding to internal repetitions within each of two or 144
more experiments; it allows for inter-experiment variation in the absolute values 145
obtained, and it identifies those differences between conditions that are consistent across 146
experiments. The number of independent experiments used for statistical analysis and 147
summarized in the graphs shown is indicated in each figure legend; the number of 148
internal repetitions was usually 3 and in some cases 2. For graphical presentation 149
purposes, some data were normalized over responses to alum or pLL; however 150
statistical analyses were always carried out on the crude data. Significances are given 151
represented as: (*) p<0.05, (**) p<0.01, and (***) p<0.001. 152
153
154
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RESULTS 155
pLL triggers NLRP3- and caspase 1-dependent IL-1β and IL-18 secretion in primed 156
BMDC 157
DCs and macrophages that have been primed with TLR agonists release IL-1β and IL-158
18 when subsequently exposed to alum or other crystalline materials (1, 2). To find out 159
if pLL could similarly trigger IL-1β and IL-18 release, we exposed LPS-primed BMDC 160
to pLL, or to alum for comparison purposes. pLL induced IL-1β and IL-18 secretion at 161
levels within the same order of magnitude to those elicited by alum (Figure 1a, b). 162
Either insoluble stimulus induced much less IL-1 than ATP (2 mM), a very potent, 163
soluble activator of the NLRP3 inflammasome that acts via the P2X7 receptor (1, 2). 164
Negligible amounts of IL-1β or IL-18 were produced in the absence of LPS priming 165
(Figure 1a, b), consistent with the previous conclusion that pLL does not contain TLR 166
agonists and/or activate NF-κB (35, 36). Because NLRP3 inflammasome activation is 167
often accompanied by some degree of cell death (1, 2), we measured cell viability 168
following exposure to pLL or alum. Exposure to pLL (at the highest dose used) or alum 169
under the assay conditions caused cell viability to drop from ca. 85% in cells only 170
primed with LPS to ca. 60% (Suppl. Fig 1). 171
In spite of the similar responses to pLL and alum observed in BMDC, a major 172
difference between the two materials was observed upon stimulation of primed BMDM. 173
Whereas alum robustly stimulated IL-1β also in macrophages, the impact of pLL 174
appeared to be restricted to BMDC, with only a low level response in macrophages 175
(Suppl. Fig. 2). 176
Reduction of disulfides alters the physicochemical properties of the LL and weakens the 177
effects of pLL on LPS-induced signaling, costimulatory molecule and cytokine 178
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responses (35, 36, 44). Consistent with these previous findings, pLL treated for 179
disulfide reduction elicited a diminished IL-1β response in BMDC (Suppl. Fig. 3). 180
To determine if IL-1β production by pLL in primed BMDC may reflect NLRP3 181
inflammasome activation, experiments were repeated in the presence of a caspase-1 182
inhibitor (Fig. 2a) or 45 mM extracellular K+ (Fig. 2b), both known to inhibit NLRP3-183
dependent IL-1β production (1). With both treatments, IL-1β production induced by 184
pLL was strongly inhibited (Fig. 2a, b). Moreover, no induction of IL-1β secretion by 185
pLL was observed in primed BMDC deficient in NLRP3 (Fig. 2c). The results for pLL 186
matched the results obtained for alum (Fig. 2a, b, c). 187
In sum, these data provide strong evidence that pLL activates the NLRP3 188
inflammasome in primed BMDC. 189
190
pLL triggers both NLRP3- and caspase 1-dependent and –independent responses 191
Exposure to pLL alters BMDC responses to LPS: it enhances CD86 expression and IL-192
10 secretion whereas it blunts CD40 expression and IL-12/23p40 secretion, as 193
previously reported (35, 36) and confirmed here (Fig. 3a-d). We wondered if these 194
alterations were a consequence of pLL-mediated NLRP3 inflammation activation. In the 195
18-h format of these experiments (see Materials and Methods) and consistent with 196
previous data (45), LPS by itself triggered an IL-1β response, but this response was 197
much potentiated in the addition presence of pLL (Fig. 3e). In the absence of LPS, pLL 198
induced negligible IL-1β. The response to LPS plus pLL (as well as to LPS alone) was 199
reduced by the caspase-1 inhibitor (Fig. 3e). Therefore, LPS can act as a priming 200
stimulus, and pLL as a second signal, in an assay format that is not designed to 201
elicit/measure NLRP3 inflammasome activation. In contrast to IL-1β, effects of pLL on 202
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the LPS-driven expression of CD86, CD40, IL-10 and IL-12/23p40 were completely 203
unaffected by chemical inhibition of caspase-1 (Fig. 3a-d). Moreover, the effects of pLL 204
on CD86, CD40, IL-10 and IL-12/23p40 were also independent of NLRP3, in the same 205
assay format (Fig. 4a-d). 206
Of note and in agreement with previous reports (39, 46, 47), alum also inhibited CD40 207
up-regulation and IL-12/23p40 secretion and potentiated IL-10 secretion in the context 208
of LPS stimulation (Fig. 4a, c, d); these effects were also independent of NLRP3 (Fig. 209
4a, c, d). 210
In sum, the effects of pLL on the LPS-induced changes in CD40, CD86, IL-10 and IL-211
12/23p40 are independent of NLRP3 inflammasome activation. 212
213
The IL-1β responses induced by pLL require actin dynamics, Syk and PI3K signaling 214
As previously mentioned, the effects caused by pLL on the LPS-induced BMDC 215
responses in terms of CD86, CD40, IL-10 and IL-12 are abrogated by inhibitors of actin 216
dynamics or PI3K class I, as well as affected by Syk inhibitors (35, 36). Also, NLRP3-217
dependent responses to particulate stimuli have been reported to depend on actin 218
dynamics, PI3K and/or Syk (17-21, 46). Thus, we wondered, if the NLRP3-dependent 219
responses to pLL shared these mechanistic requirements. Indeed, IL-1β production 220
elicited by pLL was strongly inhibited by blockade of actin polymerization 221
(Cytochalasin D; Fig. 5a), Syk signaling (piceatannol; Fig. 5b) or a pan-PI3K inhibitor 222
(wortmannin; Fig. 5c). Moreover, inhibition of either PI3K class I (Suppl. Fig 4) or 223
PI3K class III individually led to a similar reduction in IL-1β release (Fig. 5 c). Whereas 224
IL-1β secretion in response to alum displayed similar requirements, the corresponding 225
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response to the non-particulate stimulus ATP was unaffected or only weakly inhibited 226
(Fig 5a-c). 227
We previously reported that exposure of BMDC to pLL blunts the activation of the 228
PI3K class I effector Akt elicited by disparate agonists (LPS, GM-CSF, IL-4) (36, 37). 229
This is a somewhat paradoxical effect in view of the overarching requirement for PI3K 230
class I activity for pLL to affect BMDC responses (Fig. 5c, Suppl. Fig. 4; (36)). 231
Interestingly, alum did not share the capacity of pLL to blunt Akt phosphorylation in 232
response to LPS (Suppl. Fig. 5), suggesting that BMDC responses to pLL and alum 233
have mechanistic differences, and that the effect of pLL on Akt is not a direct 234
consequence of the participation of PI3K in the overall effects of the particles. 235
In sum, induction of NLRP3-dependent IL-1β production by pLL required several 236
elements of the phagocytic machinery, and these requirements were shared by the 237
particulate stimulus alum but not by the soluble stimulus ATP. 238
239
Laminated layer particles induce IL-1β in the absence of particle phagocytosis 240
Based on existing knowledge one would assume that the requirements of actin 241
dynamics, Syk and PI3K signaling for pLL to induce IL-1β are due to pLL particles 242
needing to be internalized to trigger NLRP3 inflammasome activation. Indeed, 243
particulate NLRP3 inflammasome activators are generally thought to act via 244
phago(lyso)somal destabilization, which requires prior internalization (1, 22, 23, 48). 245
However, we have so far been unable to detect successful phagocytosis of pLL particles 246
by BMDC (unpublished observations). Further, a pLL preparation in which all particles 247
are too large for phagocytosis (pLLNP
), which we previously showed to have effects 248
similar to pLL in terms of IL-10, IL-12, CD86 and CD40 (36), also elicited a clear IL-249
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1β response (Fig 6a). This response was quantitatively weaker than the response to pLL 250
at the same dry mass dose; this is expected if the effects of pLL originate at the cell 251
surface, since at equal masses pLLNP
has less total surface area than pLL. Moreover, the 252
response to pLLNP
was sensitive to PI3K class I, PI3K class III, Syk, and actin 253
polymerization inhibitors, similar to the response to pLL (Figs. 6b and Suppl. Fig. 6), 254
suggesting that the same mechanisms are operative in both cases. 255
Thus, interaction with LL particles at the cell-surface can trigger NLRP3-dependent 256
responses, which nonetheless require elements of the phagocytic machinery. 257
258
pLL induces IL-1β in vivo 259
To verify whether activation of the NLRP3 inflammasome by particles from the LL of 260
E. granulosus may occur in vivo, pLL was injected i.p. into C57BL/6 mice with or 261
without co-injection of LPS (Fig. 7). As expected, LPS instillation induced detectable 262
IL-1β in the lavage fluid of treated animals. In line with our in vitro data, pLL 263
drastically enhanced release of IL-1β, confirming its capacity to act as NLRP3-264
inflammasome-trigger. In the absence of LPS co-injection, pLL induced modest but 265
significant levels of IL-1β, in line with in vivo results with known particulate NLRP3 266
inflammasome activators (14). 267
268
DISCUSSION 269
In this work we report that a non-cellular insoluble helminth-derived material (pLL) 270
elicits NLRP3 inflammasome-dependent cytokine production in primed BMDC by a 271
mechanism requiring the phagocytic machinery but not particle internalization. 272
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Together with our previous results (36) the observations in this work suggest that all the 273
responses elicited by pLL in BMDC start with a MATS-like cell surface interaction. 274
This is in contrast to Schistosoma mansoni soluble egg antigen, which activates the 275
NLRP3 inflammasome by a mechanism requiring the receptor dectin-2 and is 276
insensitive to inhibition of actin dynamics (3). 277
Downstream of the proposed MATS-like cell surface interaction, pLL elicits responses 278
that are both NLRP3/caspase 1–dependent and –independent (Figs. 2 - 4). Parallel 279
NLRP3/caspase 1–dependent and –independent responses in DC or macrophages have 280
also been observed for crystalline particulate adjuvants (14, 49) (results for alum in the 281
present work; Fig. 4). This suggests that eliciting both NLRP3-dependent and 282
independent responses may be a general feature of materials that trigger MATS-like 283
signaling, independently of the type of material. However, some mechanistic features 284
must differ with the material eliciting the signaling, as suggested by the ability of pLL 285
but not alum to inhibit Akt activation (Suppl. Fig. 5). We previously proposed a 286
tentative mechanism to explain the paradoxical effect of pLL on Akt activation, based 287
on competition for the PI3K class I substrate phosphatidylinositol 4,5-bisphosphate 288
between the synapse with pLL and conventional PI3K-coupled receptors (36). If this 289
mechanism is correct, it may not be operative in the synapse with alum, due to 290
qualitative, or perhaps quantitative, differences with the synapse formed with pLL. 291
The IL-1β response by pLL was abrogated by PI3K class III inhibitors (Figs. 5 and 6). 292
An explanation for this observation is made difficult by the current major uncertainties 293
surrounding the mechanisms of NLRP3 inflammasome activation (23, 50). However, 294
since our results suggest PI3K class III is needed for NLRP3 inflammasome activation 295
in response to pLL/pLLNP
and alum but not to ATP (Figs. 5 and 6), the possibility 296
should be considered that this enzyme’s role is fulfilled at the synapse with particles. 297
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Neither pLL nor alum (39) appear to be internalized by DCs. Particles that cannot be 298
phagocytosed trigger “digestive exophagy”, in which the extracellular equivalent to a 299
lysosome is formed at the synapse with the particles (51), and PI3K class III is needed 300
for (conventional) phagolysosome biogenesis (52). Exophagy has been observed in 301
particular with the cell model used in our work (BMDC generated in the presence of 302
GM-CSF), in response to aggregated LDL, a probable NLRP3 inflammasome trigger 303
(53, 54). Therefore, we reason that elements of digestive exophagy may be required for 304
NLRP3 inflammasome activation by pLL and alum (and perhaps further particulates) 305
and this would explain the requirement for PI3K class III. In contrast, digestive 306
exophagy would not be needed for the NLRP3-independent responses to particulates 307
that also take place in parallel, as suggested by our previous observation that the effects 308
of pLL on LPS-elicited CD86, CD40, IL-10 and IL-12 do not require PI3K class III 309
(36). Integrating the ideas previously discussed, an interaction with MATS-like 310
requirements in the context of frustrated phagocytosis would trigger two lines of 311
signaling: PI3K class III-dependent (possibly exophagy-dependent) signaling leading to 312
NLRP3 inflammasome activation, and PI3K class III-independent signaling leading to 313
changes observed in other LPS-initiated responses; this is summarized in Suppl. Fig. 7. 314
In addition to the mucin-based aqueous gel, the E. granulosus LL contains nano-315
deposits of calcium inositol hexakisphosphate (InsP6) (55-57). BMDC responses to LL 316
materials in terms of CD86, CD40, IL-12 and IL-10 are not appreciably affected by the 317
presence or absence of this component (35). In vitro IL-1β production by BMDC was 318
also not appreciably affected by the presence or absence of calcium InsP6 (data not 319
shown). Thus, the LL mucin-based gel appears to be sufficient to trigger the proposed 320
MATS-like interaction with the BMDC surface. 321
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As mentioned, insoluble materials so far reported to activate the NLRP3 inflammasome 322
are crystalline or otherwise rigid. Accordingly, NLRP3 inflammasome activation by 323
these materials is generally accepted to be triggered by phago(lyso)somal disruption 324
after particle phagocytosis (1, 22, 23, 48). However, some authors have demonstrated 325
NLRP3 inflammasome activation by crystals immobilized to plastic or by particles too 326
large for phagocytosis (58, 59). These authors propose that a MATS-like cell surface 327
interaction may also trigger NLRP3 inflammasome activation. Our results support this 328
proposal and importantly extend NLRP3 inflammasome activation to a material that is 329
an aqueous gel, and hence soft. Materials-level features of soft non-cellular materials 330
probably determine their potentials to activate the NLRP3 inflammasome. This is 331
suggested by our observation that reduction of disulfides in pLL weakens its capacity to 332
elicit an IL-1β response (Suppl. Fig. 3). Disulfide reduction facilitates the solubilization 333
of the LL mucin meshwork upon sonication (44). We envisage that disulphide reduction 334
alters the physical properties of pLL so that it falls below a minimum level of stiffness 335
required for the MATS-like interaction. 336
The in vivo IL-1β response elicited by pLL was much enhanced by LPS, but it 337
nonetheless reached statistically significant levels in the absence of LPS co-injection 338
(Fig. 7). This is consistent with previous observations suggesting that priming signals 339
for myeloid cell responses to particulate adjuvants are ubiquitous in vivo (14). 340
Therefore, in the E. granulosus infection setting, DCs in contact with shed LL particles 341
probably generate IL-1 (and IL-18). In addition, and as suggested by our observations 342
with non-phagocytosable particles, cells in contact with the surface of the LL as such 343
may also respond with IL-1From precedents in other helminth infections, IL-1 (and 344
IL-18) could contribute to local inflammation, down-regulate Th2 responses, and/or 345
promote Th1 and Th17 responses, both of which are detectable in cystic echinococcosis 346
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(28, 60-62). Shed particles from the E. multilocularis LL (32-34) may also be NLRP3 347
inflammasome triggers and possibly contribute to the considerable inflammatory 348
response observed in human patients (63). 349
Since the survival strategy of larval E. granulosus is based on inflammatory control (25, 350
28, 63), we hypothesize that the parasite may have evolved means to curtail NLRP3 351
activation. A major secreted E. granulosus lipoprotein, “antigen B” (not contained in 352
pLL), inhibits IL-1β output in THP-1 macrophages (64), although it is not yet clear if 353
this effect involves inhibition of NLRP3 activation. 354
pLL is the first biological particulate material to be shown to activate the NLRP3 355
inflammasome independently of phagocytosis. Other non-cellular surface structures 356
shed by tissue-dwelling helminths may share this potential. In particular, it is 357
conceivable that nematode cuticles shed during moulting may be NLRP3 358
inflammasome triggers and thus contribute to local inflammation in response to these 359
helminths. 360
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Figure legends 361
Figure 1. pLL elicits IL-1β and IL-18 production in LPS-primed BMDC. BMDC 362
were primed with LPS (10 ng/ml) for 2 h or incubated in medium only, then incubated 363
for a further 3 h with medium only, pLL (at the indicated doses, given in terms of µg 364
dry mass per million cells), alum (50 µg per million cells), or ATP (2 mM). IL-1β (a) 365
and IL-18 (b) were measured in cell supernatants. Graphs show median and ranges of 3 366
(a) or 2 (b) independent experiments with internal triplicates. Data were normalized 367
over the corresponding responses to alum. Absolute median IL-1β responses to alum in 368
primed cells were 11 ng/mL (range 4 – 27 ng/mL). Absolute median IL-1β responses to 369
pLL in primed cells were 12 ng/mL (range 8-22 ng/mL) (25 µg dose), 6 ng/mL (range 370
2-13 ng/mL) (7.5 µg dose) and 3 ng/mL (range 1-5 ng/mL) (2.5 µg dose). Absolute 371
median IL-1β responses to pLL (25 µg) in non-primed cells were 0.4 ng/mL (range 0.2-372
0.4 ng/mL). Absolute median IL-18 responses to alum in primed cells were 120 pg/mL 373
(range 86-154 ng/mL) and the corresponding values for pLL (25 µg dose) were 54 374
pg/mL (range 34-75 pg/mL). Absolute median IL-18 responses to pLL in non-primed 375
cells were 3 pg/mL (range 0 – 5 pg/mL). Asterisks represent significant differences with 376
respect to the LPS-only (no second signal) condition. 377
378
Figure 2. IL-1β production in response to pLL depends on caspase-1 and NLRP3. 379
BMDC were primed with LPS (10 ng/ml) for 2 h, then incubated for a further 3 h with 380
medium only, pLL (25 µg dry mass per million cells) or alum (50 µg per million cells) 381
and IL-1β was measured in cell supernatants. Thirty minutes before addition of pLL or 382
alum, cells were exposed to the caspase-1 inhibitor Z-YVAD-FMK (a) or to 45 mM 383
KCl (b). Alternatively, cells from NLRP3+/+
and NLRP3-/-
mice were compared (c). 384
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Graphs show median and ranges of 2 (a), 4 (b) or 3 (c) independent experiments with 385
internal triplicates. Data normalized over the corresponding responses to alum in the 386
absence of inhibitors (a, b) and in NLRP3+/+
animals (c). The median absolute values of 387
responses to alum were 21 ng/mL (range 16-27 ng/mL) (a), 19 ng/mL (range 4-47 388
ng/mL) (b), and 14 ng/mL (range 3-34 ng/mL) (c). Median absolute values of responses 389
to pLL in the absence of inhibitors and in wild-type cells were 12 ng/mL (range 11-12 390
ng/mL) (a), 23 ng/mL (8-26 ng/mL) (b), and 14 ng/mL (range 6-33 ng/mL) (c). 391
Asterisks represent significant differences with respect to stimulation with the particles 392
in the presence of vehicle only (a, b) or with respect to NLRP3+/+
cells (c). 393
394
Figure 3. pLL induces caspase 1-independent responses in BMDC in parallel to 395
eliciting caspase-1-dependent IL-1β. BMDC were exposed to pLL (25 µg dry mass 396
per million cells) or medium only, and 1 h later stimulated with LPS (10 ng/ml) or 397
added medium only; the experiment was carried out in the presence or absence of the 398
caspase-1 inhibitor Z-YVAD-FMK. Eighteen h later, expression of CD40 (a) and CD86 399
(b) at the cell surface, and IL-10 (c) and IL-12/23p40 (d) and IL-1β (e) in supernatants 400
were measured. Graphs show median and ranges of 2 independent experiments with 401
internal duplicates. Data were normalized over the corresponding responses to LPS as 402
sole stimulus. The median absolute values of responses to LPS alone were 0.4 ng/mL 403
(range 0.2 – 0.6 ng/mL) for IL-10, 5 μg/mL (range 0.4 – 9 µg/mL) for IL-12/23p40, and 404
6 ng/mL (range 5 – 7 ng/mL) for IL-1β. The median absolute values of responses to 405
pLL + LPS in the absence of inhibitor were 1.3 ng/mL (range 0.5 – 2.1 ng/mL) for IL-406
10, 1.4 μg/mL (range 0.2 – 2.6 µg/mL) for IL-12/23p40, and 20 ng/mL (range 17– 23 407
ng/mL) for IL-1β. Asterisks not associated with connecting lines represent significant 408
differences with cells exposed to vehicle only. Note that only in terms of IL-1β output 409
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were significant differences detected between conditions in the presence vs absence of 410
the inhibitor. 411
412
Figure 4. pLL and alum both elicit NLRP3-independent cellular responses. BMDC 413
from either NLRP3+/+
or NLRP3-/-
mice were exposed to pLL (25 µg dry mass per 414
million cells), alum (25 µg per million cells) or medium only, and 1 h later stimulated 415
with LPS (10 ng/ml) or added medium only. Eighteen h later, expression of CD40 (a) 416
and CD86 (b) at the cell surface were measured by flow cytometry, and IL-10 (c) and 417
IL-12/23p40 (d) in supernatants were quantitated by ELISA. Graphs show median and 418
ranges of 3 independent experiments with internal triplicates. Data were normalized 419
over the corresponding responses of NLRP3+/+
cells to LPS as sole stimulus. The 420
median absolute values of responses to LPS alone in wild-type cells were 1.3 ng/mL 421
(range 0.9-1.7 ng/mL) for IL-10 and 0.6 μg/mL (range 0.3-0.9 g/mL) for IL-12/23p40. 422
Note that no significant differences were detected between conditions in the presence vs 423
absence of the inhibitor. 424
425
Figure 5. Induction of IL-1β production by pLL or alum requires components of 426
the phagocytic machinery. BMDC were primed with LPS (10 ng/ml) for 2 h, then 427
incubated for a further 3 h with medium only, pLL (25 µg of dry mass per million cells), 428
alum (50 µg per million cells), or ATP (2 mM), and IL-1β was measured in 429
supernatants. Thirty minutes before the second signal, cells were exposed to inhibitors 430
of actin dynamics (a), Syk (b) or PI3K enzymes (c). Graphs show median and ranges of 431
2 (a, b) or 3 (c) independent experiments with internal triplicates or duplicates. Data 432
were normalized over the corresponding responses to alum in the absence of inhibitors. 433
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The median absolute values of responses to alum in the absence of inhibitors were 39 434
ng/mL (range 32-45 ng/mL) (a), 19 ng/mL (range 17-22 ng/mL) and 8 ng/mL (range 5-435
11 ng/mL). The median absolute values of responses to pLL in the absence of inhibitors 436
were 20 ng/mL (17-23 ng/mL) (a), 23 ng/mL (range 21-24 ng/mL) (b), and 3 ng/ml 437
(range 3-7 ng/mL) (c). Asterisks represent significant differences with respect to the 438
corresponding conditions in the absence of inhibitors. 439
440
Figure 6. IL-1β production can be induced by pLL in the absence of phagocytosis. 441
BMDC were primed with LPS (10 ng/ml) for 2 h, then incubated for a further 3 h with 442
medium only, pLL, or pLLNP
(at the indicated doses, in µg dry mass per million cells), 443
and IL-1β was measured in supernatants. Different doses of pLLNP
were assayed in 444
parallel to pLL (a), or cells were exposed (30 minutes before pLLNP
) to the pan-PI3K 445
inhibitor wortmannin, the PI3K class I-specific inhibitor GDC-0941, the PI3K class III-446
specific inhibitors Vps34-IN1 and SAR405, or the Syk inhibitor piceatannol (b). Graphs 447
show median and ranges of 3 (a) or 2 (b) independent experiments with internal 448
triplicates. Data were normalized over responses to pLL (25 µg). Median absolute 449
responses to pLL (25 µg) and pLLNP
(75 µg) in the absence of inhibitors were 16 ng/mL 450
(range 8 - 22 ng/mL) and 5 ng/mL (range 2 - 8 ng/mL) respectively. Asterisks not 451
associated with connecting lines represent significant differences with cells stimulated 452
with LPS only. 453
454
Figure 7. pLL elicits IL-1β in vivo. C57BL/6 mice were injected i.p. with vehicle 455
alone (PBS), pLL (150 µg dry mass per mouse), LPS (15 µg per mouse) or pLL + LPS. 456
Three h later, IL-1β was measured in the peritoneal lavage fluid. The graph shows 457
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median and data individual mice pooled from two independent experiments (n = 3 and n 458
= 5). Statistical significances were estimated by a two-way method (specified in the 459
Materials and Methods section), in which the fact that the data arise from two separate 460
experiments is taken into account. 461
462
463
464
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ACKNOWLEDGEMENTS 465
This work was funded by Wellcome Trust Project Grant 092752 (to AD and JEA) and 466
CSIC Grupos project No 977 (to AD together with A.M. Ferreira). AP was supported by 467
studentships from ANII and CAP. The authors are very grateful to M.Sc. Carlos 468
González (Montevideo, Uruguay) for his expert statistical advice. They are also grateful 469
to Yamila Martínez for her participation in the preparation of pLLNP
. 470
471
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