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Tuning Innate Immune Activation by Surface
Texturing of Polymer Microparticles: The
Role of Shape in Inflammasome Activation
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of June 17, 2017.
Christine A. Vaine, Milan K. Patel, Jintao Zhu, Eunji Lee,
Robert W. Finberg, Ryan C. Hayward and Evelyn A.
Kurt-Jones
J Immunol published online 20 February 2013
http://www.jimmunol.org/content/early/2013/02/24/jimmun
ol.1200492
http://www.jimmunol.org/content/suppl/2013/02/20/jimmunol.120049
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Supplementary
Material
Published February 25, 2013, doi:10.4049/jimmunol.1200492
The Journal of Immunology
Tuning Innate Immune Activation by Surface Texturing of
Polymer Microparticles: The Role of Shape in Inflammasome
Activation
Christine A. Vaine,*,1 Milan K. Patel,*,1 Jintao Zhu,† Eunji Lee,† Robert W. Finberg,*
Ryan C. Hayward,† and Evelyn A. Kurt-Jones*
T
he human immune system is poised to recognize and respond to foreign particulate substances including pollen,
bacteria, fungal spores, and inorganic substances. The
initial step to immune cell activation for most particulate materials is
phagocytosis, a receptor-mediated, actin-dependent process carried
out by a specialized subset of cells termed “professional phagocytes,” including neutrophils, monocytes, macrophages, and dendritic cells (1). Phagocytosis can occur through association with one
of several different cell surface proteins, including complement
receptors, FcRs, scavenger receptors, and pathogen-specific receptors, such as TLRs, mannose receptors, and lectins (2, 3). Phagocytosis by immune cells can also be used for a variety of therapeutic
*Department of Medicine, University of Massachusetts Medical School, Worcester,
MA 01605; and †Department of Polymer Science and Engineering, University of
Massachusetts Amherst, Amherst, MA 01003
1
C.A.V. and M.K.P. contributed equally to the project.
Received for publication February 8, 2012. Accepted for publication January 20,
2013.
This work was supported by National Institutes of Health Grant P01 AI083215-01
(to E.A.K.-J.) and National Science Foundation Grants CBET-0741885 and CBET0931616 (to R.C.H.). Core resources supported by Diabetes Endocrinology Research
Center Grant DK32520 also were used.
Address correspondence and reprint requests to Dr. Evelyn A. Kurt-Jones, Department of Medicine, University of Massachusetts Medical School, Lazare Research
Building, Room 226, 364 Plantation Street, Worcester, MA 01605. E-mail address:
[email protected]
The online version of this article contains supplemental material.
Abbreviations used in this article: ASC, apoptosis-associated speck-like protein;
CASP1, caspase-1; CTxB, cholera toxin subunit B; Cyt. D, cytochalasin D; dA:dT,
poly(deoxyadenylic-deoxythymidylic) acid; EDC, N-(3-dimethylaminopropyl)-N9ethylcarbodiimide hydrochloride; KO, knockout; Lat. A, latrunculin A; NLRP3,
nucleotide-binding oligomerization domain 3, leucine-rich repeat and pyrin domain
containing protein 3; PS-PEO, polystyrene-block-poly(ethylene oxide); ScEM, scanning electron microscopy; TEM, transmission electron microscopy; WT, wild-type.
Copyright Ó 2013 by The American Association of Immunologists, Inc. 0022-1767/13/$16.00
www.jimmunol.org/cgi/doi/10.4049/jimmunol.1200492
applications including vaccine adjuvants and drug delivery, using
particulate material like alum and polymeric microparticles, as reviewed in Refs. 4–6.
Although the process of phagocytosis has been extensively
studied (3, 7–14), the physical and chemical characteristics that
determine the response elicited by different particles remains
unclear. Recent pioneering work from Mitragotri’s group (15) and
subsequent studies (16–19) have demonstrated that the shape of
a polymer microparticle has a dramatic effect on phagocytosis.
Specifically, the local curvature of the particle surface that is first
encountered by the phagocyte dictates whether the actin cup
necessary to engulf the particle can be formed (15, 17) and thus
whether the particle is internalized. This suggests that immune
cells use surface curvature as a locally accessible proxy for overall
particle dimension. Other work has demonstrated that for spherical microparticles, size plays a role in uptake efficiency, with
maximal phagocytosis for particle diameters of 1–3 mm (19–21).
These findings were not dependent on the receptor used to initially
mediate attachment or internalization, pointing to a highly universal role of particle geometry in phagocytic uptake.
Following phagocytosis, particulate material such as alum, silica, asbestos, monosodium urate crystals, cobalt/chromium metal
alloys, and titanium particles are known to activate nucleotidebinding oligomerization domain, leucine-rich repeat and pyrin
domain containing protein 3 (NLRP3), a NOD-like receptor protein
located in the cytosol of macrophages (22–29). The process of
NLRP3 inflammasome activation involves a conformational change
in NLRP3 into its active form, which then associates with its adaptor protein apoptosis-associated speck-like protein (ASC) (30)
through Pyrin domain interactions. This complex leads to the recruitment of pro–caspase-1 (CASP1) through caspase activation
and recruitment domain (also known as CARD) interactions (31–
33). Pro-CASP1 is then cleaved into its active form, CASP1. Active CASP1 cleaves pro–IL-1b into its active secreted form, IL-1b.
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Polymeric microparticles have been widely investigated as platforms for delivery of drugs, vaccines, and imaging contrast agents
and are increasingly used in a variety of clinical applications. Microparticles activate the inflammasome complex and induce the
processing and secretion of IL-1b, a key innate immune cytokine. Recent work suggests that although receptors are clearly
important for particle phagocytosis, other physical characteristics, especially shape, play an important role in the way microparticles activate cells. We examined the role of particle surface texturing not only on uptake efficiency but also on the subsequent
immune cell activation of the inflammasome. Using a method based on emulsion processing of amphiphilic block copolymers, we
prepared microparticles with similar overall sizes and surface chemistries but having either smooth or highly microtextured
surfaces. In vivo, textured (budding) particles induced more rapid neutrophil recruitment to the injection site. In vitro, budding
particles were more readily phagocytosed than smooth particles and induced more lipid raft recruitment to the phagosome.
Remarkably, budding particles also induced stronger IL-1b secretion than smooth particles through activation of the NLRP3
inflammasome. These findings demonstrate a pronounced role of particle surface topography in immune cell activation, suggesting that
shape is a major determinant of inflammasome activation. The Journal of Immunology, 2013, 190: 000–000.
2
PARTICULATE SURFACE TEXTURE AND IMMUNE ACTIVATION
Materials and Methods
Microparticle preparation
Generation of solvent-in-water emulsion droplets of well-controlled sizes
via flow-focusing and conversion to particle suspensions were conducted as
described previously (37). The resulting suspensions of polystyrene-blockpoly(ethylene oxide) (PS-PEO) microparticles were dialyzed against
deionized water for 2–3 d to remove glycerol and residual chloroform and
then centrifuged and resuspended in fresh deionized water five to eight
times to remove excess and weakly adsorbed poly(vinyl alcohol) surfactant. Budding and spherical particles were ∼7–8 mm in diameter. Stock
solution concentrations of particles were ∼1.45 3 107 particles/ml.
PEO functionalization of small particles
PEO-coated particles with diameters of 0.5 and 1.0 mm were prepared by
modifying carboxyl-functionalized PS particles (Polysciences, Warrington,
PA) with amino-PEO (a-aminoethyl, v-methoxy PEO, 10 kDa; Nanocs,
New York, NY). Briefly, PS-COOH particles (0.86 mmol COOH groups, in
500 ml) were allowed to react with 6 mmol N-(3-dimethylaminopropyl)N9-ethylcarbodiimide hydrochloride (EDC), and 9.5 mmol N-hydroxylsuccinimide in an aqueous 100 mM MES buffer (pH 6) at 4˚C. Solutions
of EDC and N-hydroxylsuccinimide were freshly prepared. After 1 h at
room temperature, activated PS particles were washed twice with an MES
solution (pH 6) via centrifugation and redispersion. Next, an excess of
amino-PEO (1.29 mmol) in 2.58 ml PBS (pH 7.2) was added, followed by
incubation for 1 h at room temperature and then washing twice with PBS
via centrifugation and redispersion. Successful functionalization was confirmed by X-ray photoelectron spectroscopy on a sample of particles deposited on a silicon wafer. The particles were stored in aqueous suspension
at 4˚C until use. EDC, MES, and PBS were obtained from Sigma-Aldrich
(St. Louis, MO). The stock solution concentrations of the small spherical
particles were ∼1 3 1011 particles/ml.
Electron microscopy
Microparticle morphologies were observed by scanning electron microscopy (ScEM) and transmission electron microscopy (TEM). For ScEM,
a droplet of aqueous dispersion of particles was allowed to dry on a clean
silicon wafer, followed by coating with a thin layer of gold. Samples were
imaged using a JEOL 6320 FXV ScEM at an accelerating voltage of 10 kV.
For TEM, a droplet of particle dispersion was allowed to dry on a copper
grid coated with a carbon film (Electron Microscopy Sciences) and imaged
with a JEOL 2000 FX electron microscope operated at 200 kV.
Cell culture
Immortalized mouse macrophages from wild-type (WT), NLRP3-deficient,
ASC-deficient, and CASP1-deficient mice were provided by K. Fitzgerald
and E. Latz (University of Massachusetts Medical School, Worcester, MA)
and were generated as previously described (22) using a J2 recombinant
retrovirus carrying v-myc and v-raf(mil) oncogenes. Cells were grown in
DMEM supplemented with 10% FCS, 1% L-glutamine, and 1% penicillin/
streptomycin at 37˚C with 5% CO2. Cells were plated in GM-CSF (1 ng/
ml; eBioscience)-containing medium for 18 h prior to stimulations.
Cell stimulations
Mouse macrophages (4–5 3 105) were primed for 3 h with LPS (100 ng/ml;
Sigma-Aldrich) to upregulate pro–IL-1b expression or left unprimed (media) and then stimulated with microparticles (budding, spherical, or small
particles) at given particle-to-cell ratios (particle number:cell number),
130 mg/ml alum (Thermo Scientific), 5 mM nigericin (Sigma-Aldrich),
or transfected with 400 ng poly(deoxyadenylic-deoxythymidylic) acid
(dA:dT) (Sigma-Aldrich) using GeneJuice (EMD Chemicals) for an additional 6 or 18 h. Where indicated, cells were treated with 50 mM cathepsin B inhibitor, CA-074-Me (EMD Millipore), 250 nM latrunculin
A (Lat. A) (Sigma-Aldrich), or 1 mM cytochalasin D (Cyt. D) (SigmaAldrich). Secreted IL-1b was measured using ELISA (R&D Systems),
according to the manufacturer’s instructions.
Confocal microscopy
Cells were cultured on glass-bottom 35-mm tissue-culture dishes (MatTek)
in complete medium. Where indicated, cells were stained with LysoTracker
Green, Hoechst 34580, and Alexa 488–cholera toxin subunit B (CTxB)
from Molecular Probes (Invitrogen), according to the manufacturer’s
instructions. Images were taken on a Leica SP2 Acousto Optical Beam
Splitter confocal laser-scanning microscope with a 363 objective, using
Leica Confocal Software. Multicolor images were acquired by sequential
scanning with only one laser active per scan to avoid cross-excitation.
Overall brightness and contrast of images were optimized using Adobe
Photoshop CS3.
Mice injections
C57BL/6 (WT), IL-1R–knockout (IL-1R KO), and CASP1KO mice were
obtained from The Jackson Laboratory (Bar Harbor, ME). NLRP3KO mice
were provided by K. Fitzgerald (University of Massachusetts Medical
School). Mice were injected i.p. with sterile PBS (400 ml), 4% thioglycollate
(1 ml), or 2 3 10 6 microparticles (∼450 mg). Mice were sacrificed by
isoflurane inhalation, followed by cervical dislocation. Peritoneal exudate
cells (PECs) were isolated 6 or 16 h after injections as described previously
(29). All mouse strains, age- and sex-matched with appropriate controls,
were bred and maintained at the University of Massachusetts Medical
School animal facility. Experiments involving live animals were in accordance with guidelines set forth by the University of Massachusetts
Medical School Department of Animal Medicine and the Institutional
Animal Care and Use Committee.
Flow cytometric analysis
Neutrophils in PECs were enumerated as described previously (29). Data
were acquired by DIVA (BD Biosciences) and were analyzed with FlowJo
8.8.6 software (Tree Star).
Statistical analysis
An unpaired, two-tailed Student t test was used to determine statistical
significance of independent experiments where two groups were compared When more than two groups were compared, ANOVA followed by
Bonferroni’s correction for posttest comparisons was used. Values of p ,
0.05 were considered significant with 95% confidence intervals. Statistics
were performed using GraphPad (Prism version 5.0d) software.
Results
Preparation of budding particles
PS-PEO particles are an excellent model system as PS-PEO has
been well studied in the context of generation of textured particles
(37). There have also been several studies examining the use of PS
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IL-1 is a potent inflammatory cytokine, important for functions
including macrophage and neutrophil recruitment, as well as T cell
activation (34, 35).
Although a striking correlation between geometry and engulfment
rate has been established for “simple” particle shapes (i.e., spheres,
ellipsoids, and discs) (15–19, 28), the potential to use more complex particle shapes to engineer the phagocytic response remains
largely untapped. Furthermore, questions of how particle geometry
dictates the immune response following phagocytosis, such as IL1b release, have not been addressed. Our understanding of these
effects remains fairly limited, primarily because until recently,
methods did not exist to produce uniform microparticles with controlled surface chemistry and systematically varying shapes.
In this study, we take advantage of a recently developed route to
prepare polymeric microparticles with complex but well-controlled
surface topographies, based on emulsion processing of amphiphilic
block copolymers (36, 37). This provides a simple platform to
compare the response of phagocytes to particles of similar overall
size and surface chemistry but where the particle surfaces are
either smooth or densely covered with microscale protrusions
(which we refer to as textured or “budding” particles). In this way,
we can assess the role of shape independent of receptor interaction
with particles based on their surface chemistry. Because phagocytes respond to local surface curvature (15–19, 28), we anticipated that the regions of high curvature on the budding particles
should substantially alter the immune response. We examined the
acute inflammatory response to polymeric microparticles via
neutrophil recruitment in an in vivo mouse peritonitis model. We
further analyzed the mechanism of this response using mouse
macrophages to compare the ability of smooth and budding particles
to be phagocytosed, activate immune cells, activate the inflammasome, and induce IL-1b cytokine release.
The Journal of Immunology
3
and PS-PEO particles as therapeutic agents (15, 38, 39). Therefore, to study the role of surface texturing on the immune response, we prepared polymer microparticles consisting of PS-PEO
diblock copolymer (38 and 11 kg/mol number-average molecular
weights Mn, respectively) blended with PS homopolymer (Mn =
12.4 kg/mol). Initially, the polymers are dissolved in chloroform
and then emulsified in water using a microfluidic flow-focusing
device to provide droplets of uniform size (37). Removal of the
organic solvent by evaporation subsequently yields solid polymer
microparticles of phagocytosable size (7–8 mm diameter). As
shown in Fig. 1, by adjusting the mass ratio of PS-PEO:PS, the
morphology of the particles can be changed from budding particles (100:0; Fig. 1A), which are densely coated with vesicular
protrusions of 1–2 mm diameter, to smooth spherical particles
(20:80; Fig. 1B). Because of the interfacial activity of PS-PEO,
the surfaces of both types of particles are coated with a similar
“brush” layer of PEO as well as residual poly(vinyl alcohol) used
to stabilize the emulsion droplets.
We first compared the in vivo immune response to budding particles
versus spherical particles. Previous studies have shown that i.p.
injections of particulates (e.g., monosodium urate crystals or titanium
particles) lead to an increase in neutrophil recruitment (23, 29). To
determine whether our polymer microparticles induced a similar
increase in neutrophil recruitment, WT mice were first injected i.p.
with budding or spherical particles at three different doses: ∼6.7 3
105, 2 3 106, and 2.7 3 106 particles (150, 450, and 600 mg, respectively), and lavage of the peritoneal cavity was analyzed for
neutrophil influx 6 h later. We found that the 450-mg dose of budding particles induced significantly higher neutrophil recruitment
(Ly6G+, 7/4+ cells) over spherical particles (p , 0.01, budding
versus spherical; Fig. 2A). In fact, 450 mg budding particles induced
neutrophil recruitment at levels similar to injection with the positive
control, thioglycollate (p , 0.001, PBS versus thioglycollate; Fig.
2A). Next, we compared the neutrophil recruitment to 450 mg
particles at 6 or 16 h after injection. Both budding and spherical
particles induced a significant neutrophil response over PBS-carrier
only at 6 h (p , 0.0001, both) and 16h (p , 0.01, budding; p ,
0.0001, spherical) (Fig. 2 B). Budding particles exhibited significantly higher levels of neutrophil recruitment than spherical particles at 6 h but only slightly higher levels at 16 h when compared
with spherical particles (Fig. 2A, 2B).
Microparticle-induced neutrophil recruitment involves
IL-1–associated signaling
The IL-1R is required for neutrophil recruitment following exposure to stimulants/particulates in mice (22, 23, 29, 40). To determine whether the IL-1R was required for microparticle-induced
neutrophil influx, IL-1R KO mice and WT mice were injected
with budding or spherical particles for 16 h and compared with
FIGURE 2. Particle-induced neutrophil recruitment depends on surface
curvature and requires IL-1R and NLRP3 inflammasome–associated signaling. Flow cytometric analysis on peritoneal neutrophil (Ly6G+, 7/4+
cells) recruitment at 6 h (A, D, E), 16 h (C), or at given times (B) after
microparticle injections at varying doses as indicated (A) or at a fixed dose
of 2 3 106 particles; ∼450 mg (B, C, D, E) in WT (C57BL/6), IL-1RKO,
NLRP3KO, or CASP1KO mice. Graphs show mean + SEM of total
number of mice indicated below, performed in two to three independent
experiments. Y-axis scales are (3106) [(A)–(D)] or (3105) (E). Number of
mice: (A) PBS (0), n = 4; 150 mg, n = 2; 450 mg, n = 6 (spherical) and 8
(budding); 600 mg, n = 2 (spherical) and 3 (budding); thioglycollate (Thio),
n = 3. (B) 0, n = 9; 6B, n = 8; 6S, n = 6; 16B, n = 5; 16S, n = 5. (C) PBS, n =
5; WT, n = 5; KO, n = 3. (D) n = 2. Significance values are shown as
budding versus spherical or Thio versus PBS (A), particle versus PBS
injections (B), PBS versus particle injections (C), or KO versus WT (D).
*p , 0.05, **p , 0.01, ***p , 0.001, ****p , 0.0001.
PBS alone injections. As predicted, IL-1R KO mice did not recruit
neutrophils in response to particles over PBS alone, whereas WT
mice exhibited a significant increase over PBS (p , 0.05, budding; p , 0.001, spherical; Fig. 2C).
NLRP3 inflammasome is critical for microparticle-induced
neutrophil recruitment
FIGURE 1. Images of budding and spherical microparticles. ScEM and
TEM (inset images) images of budding (A) and spherical (B) particles
generated. Scale bars, 10 and 5 mm (inset).
The NLRP3 inflammasome plays a role in the response to particulate materials (22–29) through IL-1R and IL-1b secretion. To
determine whether components of the NLRP3 inflammasome were
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Budding particles stimulate a more robust neutrophil response
at early time points in vivo
4
PARTICULATE SURFACE TEXTURE AND IMMUNE ACTIVATION
required for particle-induced neutrophil recruitment, we examined
peritoneal neutrophil recruitment to budding particles in NLRP3 KO
and CASP1 KO mice and to spherical particles in NLRP3 KO mice
6 h following particle injections. When budding particles were
injected, we found that the NLRP3 KO and CASP1 KO mice exhibited significantly blunted neutrophil recruitment, compared with
WT controls in response to budding particles (p , 0.05, WT versus
all KOs; Fig. 2D). Again, spherical particles induced substantially
less neutrophil recruitment than budding particles (Fig. 2E). The
neutrophil response to spherical particles was also inhibited in
NLRP3 KO compared with WT mice, although trending did not
reach statistical significance with a p value of 0.0674.
Budding particles stimulate more IL-1b than spherical
particles
Microparticle-induced IL-1b production requires the NLRP3
inflammasome
To further determine whether the NLRP3 inflammasome signaling
complex was involved in the IL-1b response to microparticles in
macrophages, immortalized macrophages generated from NLRP3
KO, ASC KO, and CASP1 KO mice were analyzed for IL-1b
production in response to microparticle stimulation. Supernatants
from NLRP3 KO, ASC KO, and CASP1 KO macrophages each
exhibited undetectable levels of secreted IL-b in response to budding and spherical microparticles, whereas IL-1b secretion from
WT macrophages was readily detected (p , 0.0001, WT versus all
KOs; Fig. 4A). As expected, NLRP3 KO macrophages could respond to dA:dT similar to WT cells, whereas ASC KO and CASP1
KO macrophages could not (p , 0.01, NLRP3 versus WT; p ,
0.0001, ASC/CASP1 KO versus WT; Fig. 4B). WT and deficient
cells also produced equivalent levels of the inflammasome-independent cytokine, IL-6 (Supplemental Fig. 1B), following LPS
stimulation.
FIGURE 3. Particle-induced IL-1b cytokine secretion is dependent on
surface curvature. Secreted IL-1b levels from WT immortalized mouse
macrophages. Cells were incubated with media alone (media) or primed
with LPS for 3 h and then stimulated with budding or spherical particles
with increasing particle-to-cell ratios (particle number:cell number; 1:1,
∼100 mg) (A) or transfected with 400 ng dA:dT (B) for 6 h. Kinetics of IL1b secretion from 100 mg budding or spherical particles (C). Cytokine
levels are reported as mean + SEM and are representative of two independent experiments performed in duplicate. Significance values are
shown as budding versus spherical (A, C) or prime versus dA:dT (B). *p ,
0.05, **p , 0.01, ****p , 0.0001.
Small, spherical particles do not induce significant IL-1b
secretion
Studies have shown that particles within the range of 1–3 mm in
diameter exhibit the highest phagocytic rates when incubated with
macrophages (19–21). The “buds” on the budding particles are
∼1–2 mm in diameter. To determine whether the presence of these
small buds is responsible for the increased IL-1b secretion exhibited by budding particles when compared with spherical particles, we also tested the response to smaller spherical particles
with diameters of 0.5 and 1 mm, the surfaces of which were also
coated with PEO. These smaller spherical particles were incubated
with WT immortalized macrophages and assayed for IL-1b secretion. Unlike the budding particles, these small (bud-sized)
spherical particles did not induce a significant amount of IL-1b
secretion above prime-only background values (Fig. 5), even at
high particle-to-cell ratios. All samples exhibited similar levels of
NLRP3-independent cytokine IL-6 (Supplemental Fig. 1C) following LPS prime. Of note, uncoated 0.5- and 1-mm PS particles
were also unable to induce significant IL-1b secretion over primeonly levels (data not shown).
Budding particles are more likely to be associated with and
internalized in mouse macrophages than spherical particles
Because inflammasome activation of IL-1b release generally
involves phagocytosis of the stimulant, we examined the ability of
macrophages to attach to and internalize spherical and budding
particles. To visualize phagocytosis of microparticles, immortalized macrophages from WT mice were incubated for 6 h with
budding or spherical microparticles containing a fluorescent dye,
Vibrant DiI. Using confocal microscopy, we found that a significantly higher percentage of budding particles (.85%) bound to or
internalized in macrophages (Fig. 6A, 6C, 6D) when compared
with spherical particles, where only ∼20% were bound or inter-
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To determine whether the observed IL-1R– and NLRP3 inflammasome–dependent neutrophil influx in vivo exhibited similar
characteristics in vitro, we analyzed the IL-1b inflammasome response of murine macrophages to microparticles. Immortalized
macrophages from WT mice were primed for 3 h with LPS to
induce upregulation of pro–IL-1b transcription and then incubated
with budding or spherical microparticles for 6 h to induce pro–IL1b processing to mature IL-1b. Levels of mature, secreted IL-1b
were measured via ELISA using cell supernatants. As predicted by
the neutrophil recruitment studies, budding particles were able to
induce significantly higher levels of IL-1b secretion (p , 0.0001,
0.5:1 and 1:1; Fig. 3A, n) compared with spherical particles (Fig.
3A, N). WT macrophages responded to both types of microparticles
in a dose-dependent manner with significantly higher levels of
secreted IL-1b compared with priming alone at the 1:1 particle:cell
ratio (4.5 3 105 particles) for both types of particles as well as the
0.5:1 particle:cell ratio (2.25 3 105 particles) for budding particles
(Fig. 3A). As a positive control, macrophages were transfected
with synthetic dsDNA, dA:dT. WT macrophages produced a
significant amount of IL-1b in response to dA:dT stimulation
(p , 0.01; Fig. 3B). As negative controls, supernatants from macrophages that received media or an LPS prime alone did not exhibit an increase in IL-1b production. Levels of NLRP3-independent cytokine IL-6 were equivalent for all samples (Supplemental
Fig. 1A). Because the neutrophil response seen with budding
particles was similar to spherical particles at 16 h in vivo, we also
examined the in vitro kinetics of the IL-1b response to microparticles. Unlike neutrophil recruitment, budding particles stimulated higher levels of IL-1b at all time points compared with
spherical particles (p , 0.01, 6 h; p , 0.05, 16 h; Fig. 3C).
The Journal of Immunology
5
nalized (p , 0.0001, budding versus spherical; Fig. 6B, 6D). We
also found that budding particles were associated with more
macrophages on a per particle basis, with the majority of budding
particles associated with two to three macrophages each. In other
words, a single budding particle was often associated with more
than one macrophage at a time (Fig. 6C, 6E). In contrast, a single
spherical particle only associated with a single macrophage (Fig.
6B, 6E). These data are trending toward significance with a p
value of 0.0632. As internal controls, the total number of cells per
field of view and number of particles per field of view were very
similar between spherical and budding particles, whereas the absolute number of bound budding particles was still significantly
higher than bound spherical particles (p , 0.01, bound budding
versus spherical; Supplemental Fig. 2A–C).
FIGURE 5. Small spherical particles do not induce IL-1b secretion.
Secreted IL-1b levels from WT immortalized mouse macrophages. Cells
were incubated with media alone (media) or primed with LPS for 3 h
(prime) and then stimulated with small spherical particles (0.5 or 1 mm
diameter) for 6 h with increasing particle-to-cell ratios (particle number:
cell number; 1:1, ∼100 mg). Cytokine levels are reported as mean + SEM
and are representative of two independent experiments performed in
duplicate.
FIGURE 6. Particle phagocytosis: budding particles associate with more
macrophages than spherical particles. Confocal microscopy images of
macrophage-associated budding (A, C) and spherical (B) particles following a 3-h prime with LPS and 6-h incubation with particles. Lysosomes
were visualized with LysoTracker Green. Nuclei were visualized with
Hoechst 34580 (blue). Scale bars, 10 mm. Images were taken with a 363
objective. Percentage of and average (line) particles bound to macrophages
per field of view (D). Total and average (indicated by line) number of
macrophages associated with a single particle per field of view (E).
Analysis was performed on seven independent fields of view per particle
type. Secreted IL-1b from WT immortalized macrophages stimulated for
6 h (F) or 18 h (G) with 100 mg budding or spherical particles, 130 mg/ml
alum, or 5 mM nigericin in the presence (N) or absence (n) of 50 mM CA074-Me (F), 250 nM Lat. A, or 1 mM Cyt. D (G). Cytokine levels are
reported as mean + SEM and are representative of two independent
experiments performed in duplicate. Significance values are shown as
budding versus spherical particles (D) or untreated versus treated cells (F,
G). **p , 0.01, ****p , 0.0001.
Optimal inflammasome activation in response to silica crystals,
alum, amyloid-b, and titanium requires uptake through actin polymerization and release of cathepsin B following lysosomal destabilization (22, 29, 41). To determine whether actin polymerization
and cathepsin B are required for inflammasome activation and
subsequent IL-1b production in response to microparticles, WT
immortalized mouse macrophages were treated with the cathepsin B
inhibitor CA-074-Me or actin inhibitors Lat. A and Cyt. D. Supernatants from cells pretreated with CA-074-Me had substantially
lower levels of IL-1b following a 6-h microparticle stimulation
compared with untreated cells (p , 0.0001, untreated versus treated;
Fig. 6F). In addition, cells pretreated with Lat. A or Cyt. D exhibited
a complete loss of IL-1b following an 18-h microparticle stimulation (p , 0.0001, untreated versus treated; Fig. 6G). As expected,
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FIGURE 4. Particle-induced IL-1b cytokine release requires the NLRP3
inflammasome. Secreted IL-1b levels from immortalized mouse macrophages from WT, NLRP3 KO, ASC KO, and CASP1 KO mice that were
primed for 3 h with LPS (prime) and then stimulated with 4.5 3 105 (1:1
particle:cell ratio) spherical or budding microparticles for 6 h (A) or
transfected with dA:dT (B). Cytokine levels are reported as mean + SEM
and are representative of three independent experiments performed in
duplicate. Significance values are shown as WT versus KO. **p , 0.01,
****p , 0.0001.
6
PARTICULATE SURFACE TEXTURE AND IMMUNE ACTIVATION
alum-induced IL-1b requires both cathepsin B and actin (p ,
0.0001, untreated versus treated, all; Fig. 6F, 6G), whereas nigericin,
a potassium ionophore known to induce IL-1b through potassium
efflux, lysosomal destabilization, and cathepsin B release (42), requires cathepsin B but does not require actin polymerization (p ,
0.0001, CA-074-Me treatment and p , 0.01, Lat. A and Cyt. D treatments; Fig. 6F, 6G). Furthermore, macrophages that were transfected with dsDNA, dA:dT, which induces mature IL-1b production in an NLRP3-independent manner (43), were unaffected by
cathepsin B inhibition (data not shown).
Budding particles localize with lipid raft components
Discussion
This study illustrates a significant role for surface texturing in
polymer microparticle-induced inflammasome stimulation, both
in vivo and in vitro independent of the chemical composition of the
particle. The use of macrophage cell lines allowed us to interrogate
the potential pathways triggered by particles and revealed roles for
NLRP3, ASC, CASP1, and IL-1R and suggest that budding particles induce a significantly greater inflammasome response than
spherical particles. These observations have been validated in vivo
using neutrophil recruitment, confirming the importance of these
pathways in vivo and supporting our hypothesis that surface texture
FIGURE 7. Internalized budding particles localize with lipid raft components. Confocal microscopy images of macrophage-associated spherical
(A) and budding (B) particles. Cells were incubated with particles for 6 h
and then fixed with 4% paraformaldehyde for 20 min prior to visualization.
Lipid rafts were visualized with CTxB–Alexa 488 (green). Nuclei were
visualized with Hoechst 34580 (blue). Arrows indicate localization of lipid
rafts with particle. Images are representative of two independent experiments. Scale bars, 5 mm. Images taken with a 363 objective.
FIGURE 8. Particle-induced inflammasome activation and neutrophil
recruitment. Particle internalization, via actin polymerization, triggers the
release of cathepsin B from lysosomes, which together activate NLRP3.
Activated NLRP3 recruits ASC through PYD domain interactions. This
complex triggers recruitment and cleavage of activated caspase-1 through
caspase activation and recruitment domain (also known as CARD) interactions. This inflammasome complex then cleaves pro–IL-1b into its active,
secreted form IL-1b, which can trigger downstream IL-1–associated signaling, including neutrophil recruitment, through activation of the IL-1R.
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Recruitment of lipid rafts plays an important role in phagocytosis
and inflammatory cytokine secretion by anchoring scavenger
receptors, innate immune receptors (such as CD14 and Dectin-1)
during activation of downstream kinases (44–46). In addition, we
have previously shown that uptake of Ab-bound membrane proteins can internalize via clathrin through the endosomal pathway
(47). To further elucidate the mechanisms involved in particle
internalization, WT macrophages were stained with fluorescent
CTxB, which binds to GM1 gangliosides on the cell surface, as
a marker for lipid rafts. Cells were then incubated with fluorescent
particles for 6 h. We found that the surface of budding particles
highly localized with CTxB (arrows), whereas spherical particles
did not (Fig. 7). Overall, our studies identify an important role for
surface texturing in microparticle phagocytosis and NLRP3 activation, ultimately leading to neutrophil recruitment (illustrated in
Fig. 8).
is an important determinant of inflammasome activation by particles.
We and others have demonstrated a critical role for IL-1associated signaling in neutrophil responses following injections
of particulate stimuli (22, 23, 29, 40). Here we show that IL-1R
KO mice exhibit a diminished neutrophil response following microparticle injections, further implicating IL-1 signaling in the innate immune response to budding and spherical polymer microparticles. Furthermore, we verify that this response also requires
a functional NLRP3 inflammasome, as NLRP3 KO and CASP1
KO mice were unable to recruit a significant amount of neutrophils following particle injections. It is possible that sensors in
addition to NLRP3 may play a role in particle induced neutrophil
recruitment and this may account for the lack of a complete abolition of neutrophil recruitment in KO mice. However, regardless
of whether NLRP3 is the only inflammasome receptor or one of
several inflammasome receptors that are triggered by particles,
these studies clearly demonstrate that the downstream ASC and
CASP1 pathways and the IL-1R pathway are very important for
neutrophil responses to budding and spherical particles.
We determined that although there is some variation in the
amount of IL-1b detected in macrophage supernatants, spherical
particles consistently and reproducibly induced significantly less
IL-1b than budding particles in every experiment in side-by-side
comparisons. Particle IL-1b induction also occurs through activation of the NLRP3 inflammasome–signaling complex, similar
to that seen with other particulate stimuli (22–29). Our kinetic
studies in vitro also indicate that budding particles induced an
early peak and continued high IL-1b secretion over time, whereas
spherical particles induced lower IL-1b secretion levels that remained relatively constant. In vivo, it is unclear why spherical
particles appear to “catch up” with budding particles for neutrophil recruitment at later time points. There is likely a complex
interplay between a variety of signals in vivo, including the magnitude of inflammasome activation for IL-1b secretion, levels of
IL-1b produced, IL-1b–driven neutrophil recruitment, and adherence of activated neutrophils to peritoneal tissues and/or pyroptosis/necrosis of highly activated neutrophils.
Budding particles also induced a more rapid phagocytic response
in vitro and were more readily taken up by macrophages than
spherical particles, again suggesting that the shape of the particle
The Journal of Immunology
macrophages (19), rat alveolar macrophages (20), and peritoneal
mouse macrophages (21). We have demonstrated that immortalized mouse macrophages, which have been used for inflammasome activation studies to a variety of particulate material (22,
29), appear to respond differently (and very weakly) to small,
spherical particles when compared with the response from murine
dendritic cells, peritoneal macrophages, and rat alveolar macrophages.
Overall, the data presented in this report have broad implications
for the future development of vaccine adjuvants and therapeutic
delivery agents, because variation in surface curvature will modulate
the resultant immune response. We suggest that larger biodegradable particles with low surface curvature and no complex surface
structures would be ideal for use as delivery vehicles, because they
exhibit a low level of uptake and induce a slow immune response
and, thus, may exhibit a broader biodistribution, ideal for delivery of
therapeutic agents. In contrast, we suggest that particles with high
surface curvature and complex surface structure would be ideal for
use as vaccine adjuvants, as they are more efficiently phagocytosed
in cells and induce a more robust immune response. Thus, both the
chemical composition-dependent receptor engagement and surface
texture–dependent inflammasome activation are key elements determining the uptake and immune activation potential of particles.
Acknowledgments
We thank the following people from University of Massachusetts Medical
School (Worcester, MA): Melvin Chan and Megan Munroe for technical
assistance, Melanie Trombly for critical review of the manuscript, and
Katherine Fitzgerald and Eicke Latz for immortalized macrophages.
Disclosures
The authors have no financial conflicts of interest.
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