Serum Amyloid A Activates the NLRP3 Inflammasome and Promotes Th17 Allergic Asthma in Mice

Vermont Lung Center, Division of Pulmonary Disease and Critical Care, Department of Medicine, University of Vermont, Burlington, VT 05405, USA.
The Journal of Immunology (Impact Factor: 4.92). 07/2011; 187(1):64-73. DOI: 10.4049/jimmunol.1100500
Source: PubMed
IL-1β is a cytokine critical to several inflammatory diseases in which pathogenic Th17 responses are implicated. Activation of the NLRP3 inflammasome by microbial and environmental stimuli can enable the caspase-1-dependent processing and secretion of IL-1β. The acute-phase protein serum amyloid A (SAA) is highly induced during inflammatory responses, wherein it participates in systemic modulation of innate and adaptive immune responses. Elevated levels of IL-1β, SAA, and IL-17 are present in subjects with severe allergic asthma, yet the mechanistic relationship among these mediators has yet to be identified. In this study, we demonstrate that Saa3 is expressed in the lungs of mice exposed to several mixed Th2/Th17-polarizing allergic sensitization regimens. SAA instillation into the lungs elicits robust TLR2-, MyD88-, and IL-1-dependent pulmonary neutrophilic inflammation. Furthermore, SAA drives production of IL-1α, IL-1β, IL-6, IL-23, and PGE(2), causes dendritic cell (DC) maturation, and requires TLR2, MyD88, and the NLRP3 inflammasome for secretion of IL-1β by DCs and macrophages. CD4(+) T cells polyclonally stimulated in the presence of conditioned media from SAA-exposed DCs produced IL-17, and the capacity of polyclonally stimulated splenocytes to secrete IL-17 is dependent upon IL-1, TLR2, and the NLRP3 inflammasome. Additionally, in a model of allergic airway inflammation, administration of SAA to the lungs functions as an adjuvant to sensitize mice to inhaled OVA, resulting in leukocyte influx after Ag challenge and a predominance of IL-17 production from restimulated splenocytes that is dependent upon IL-1R signaling.


Available from: Richard A Flavell
The Journal of Immunology
Serum Amyloid A Activates the NLRP3 Inflammasome and
Promotes Th17 Allergic Asthma in Mice
Jennifer L. Ather,* Karina Ckless,
Rebecca Martin,* Kathryn L. Foley,*
Benjamin T. Suratt,* Jonathan E. Boyson,
Katherine A. Fitzgerald,
Richard A. Flavell,
Stephanie C. Eisenbarth,
and Matthew E. Poynter*
IL-1b is a cytokine critical to several inflammatory diseases in which pathogenic Th17 responses are implicated. Activation of the
NLRP3 inflammasome by microbial and environmental stimuli can enable the caspase-1–dependent processing and secretion of
IL-1b. The acute-phase protein serum amyloid A (SAA) is highly induced during inflammatory responses, wherein it participates
in systemic modulation of innate and adaptive immune responses. Elevated levels of IL-1b, SAA, and IL-17 are present in subjects
with severe allergic asthma, yet the mechanistic relationship among these mediators has yet to be identified. In this study, we
demonstrate that Saa3 is expressed in the lungs of mice exposed to several mixed Th2/Th17-polarizing allergic sensitization
regimens. SAA instillation into the lungs elicits robust TLR2-, MyD88-, and IL-1–dependent pulmonary neutrophilic inflamma-
tion. Furthermore, SAA drives production of IL-1a, IL-1b, IL-6, IL-23, and PGE
, causes dendritic cell (DC) maturation, and
requires TLR2, MyD88, and the NLRP3 inflammasome for secretion of IL-1b by DCs and macrophages. CD4
T cells polyclonally
stimulated in the presence of conditioned media from SAA-exposed DCs produced IL-17, and the capacity of polyclonally
stimulated splenocytes to secrete IL-17 is dependent upon IL-1, TLR2, and the NLRP3 inflammasome. Additionally, in a model
of allergic airway inflammation, administration of SAA to the lungs functions as an adjuvant to sensitize mice to inhaled OVA,
resulting in leukocyte influx after Ag challenge and a predominance of IL-17 production from restimulated splenocytes that is
dependent upon IL-1R signaling. The Journal of Immunology, 2011, 187: 64–73.
he IL-1 family of cytokines is critical to the host response
to infection, playing a variety of functions not only in the
acute-phase response from the liver, but also in alterations
in metabolism, induction of fever, and lymphocyte activation (1).
Overproduction of IL-1b, in particular, is thought to be re-
sponsible for a variety of autoinflammatory syndromes such as
familial Mediterranean fever and Muckle-Wells syndrome and is
also a contributing factor in osteoarthritis, rheumatoid arthritis,
gout, multiple sclerosis (experimental autoimmune encephalo-
myelitis), colitis, diabetes, and Alzheimer’s disease (2–9). Setting
IL-1b apart from other acute-phase cytokines such as IL-6 and
TNF-a is the requirement for processing from an inactive proform
to an active secreted form by caspase-1 cleavage, which itself is
activated by the assembly of a cytoplasmic inflammasome com-
plex. The NLRP3 inflammasome is not only critical for IL-1b
release in response to a variety of stimuli, but has also been im-
plicated in several of the same autoimmune and autoinflammatory
disorders in which IL-1b plays a causative role. Key to these
models of inappropriate adaptive immune responsiveness is the
development of a Th17-biased phenotype, and recent research has
highlighted the importance of IL-1b in Th17 development (10,
11). IL-1b, in conjunction with IL-6 and TGF-b, is critical in
humans for the development of the Th17 lineage. Not only do
Th17 cells upregulate mRNA expression of the IL-1R compared
with Th1 and Th2 cells, but lack of this receptor on polyclonally
stimulated T cells results in significant reduction of IL-17A, IL-
17F, IL-21, and IL-22 production (10, 12).
Long known to be a biomarker of inflammation in a multitude of
diseases (13), serum amyloid A (SAA) is also a critical mediator
of disease pathogenesis. SAA can stimulate cells via TLR2 to
elicit a robust signaling cascade in human monocytes (14) and
mouse macrophages (15), whereas it can also signal through the
formyl-peptide receptor (FPR)-like 1/FPR2 to promote neutrophil
chemotaxis and activation (16–18). In addition, SAA induces
expression of matrix metalloproteinases and collagenases that are
pivotal in tissue remodeling after injury (19). SAA can also pro-
mote the development of Th17 responses, which has been dem-
onstrated to be an important mechanism by which segmented
filamentous bacteria induce intestinal disease (20). However, the
importance of SAA on modulating adaptive immune responses in
other tissues and beyond the scope of infection has not yet been
demonstrated. Most well known is the role of SAA fibril de-
position in severe conditions such as amyloidosis (21). It has re-
cently been demonstrated that b-amyloid fibrils in Alzheimer’s
disease (22) and islet amyloid polypeptide in type 2 diabetes (2)
signal through the NLRP3 inflammasome and drive caspase-1–
dependent cleavage of IL-1b.
*Vermont Lung Center, Division of Pulmonary Disease and Critical Care, Depart-
ment of Medicine, University of Vermont, Burlington, VT 05405;
Department of
Chemistry, State University of New York Plattsburgh, Plattsburgh, NY 12901;
Department of Surgery, University of Vermont, Burlington, VT 05405;
of Infectious Diseases and Immunology, Department of Medicine, University of
Massachusetts Medical School, Worchester, MA 01655;
Howard Hughes Medical
Institute, Department of Immunobiology, Yale University School of Medicine, New
Haven, CT 06520; and
Department of Laboratory Medicine, Yale University School
of Medicine, New Haven, CT 06520
Received for publication February 16, 2011. Accepted for publication April 21, 2011.
This work was supported by Grants R01 HL0 89177 and P20 RR15557 from the
National Institutes of Health.
Address correspondence and reprint requests to Dr. Matthew E. Poynter, Division of
Pulmonary Disease and Critical Care, Department of Medicine, University of Ver-
mont, Given E410A, 89 Beaumont Avenue, Burlington, VT 05405. E-mail address:
Abbreviations used in this article: Alum, aluminum hydroxide; BAL, bronchoalveo-
lar lavage; BMDC, bone marrow-derived dendritic cell; CAIKKb, constitutively
active IkB kinase b; DC, dendritic cell; Dox, doxycycline; FPR, formyl-peptide
receptor; o.a., oropharyngeal aspiration; SAA, serum amyloid A.
Copyright Ó 2011 by The American Association of Immunologists, Inc. 0022-1767/11/$16.00
Page 1
Asthma is conventionally considered to be a Th2-driven disease
associated with wheezing, airway hyperresponsiveness, IgE, eo-
sinophilia, and mucus metaplasia. However, in a substantial per-
centage of patients, asthma presents as nonatopic, instead mani-
festing as a neutrophilic and steroid-resistant phenotype that re-
sults in increased severity and morbidity of disease (23, 24).
Severe allergic asthma is associated with elevated levels of several
mediators, including SAA (25–27), IL-1b (28), and IL-17 (29–
36), although a mechanistic link among these molecules has not
yet been established. IL-1b and IL-17A have been demonstrated
to upregulate expression of the mucin gene Muc5ac (37), and
IL-1b also acts via cyclooxygenase-2 and PGE
production to
desensitize airway smooth muscle cells to b-adrenergic agonists
Mouse models of allergic asthma have classically exploited the
Th2-promoting adjuvant aluminum hydroxide (Alum), delivered
as an emulsion with Ag via i.p. injection (39). However, allergic
asthma models are evolving to encompass inhalational methods
of sensitization to aeroantigens, which promote a mixed Th2/Th17
allergic airway disease phenotype (40). Although the Th17 re-
sponse in mouse models of allergic airway disease is associated
with neutrophilia, tissue destruction, and steroid unresponsiveness,
little is known about the endogenous mediators that are critical to
this response. Recent reports have implicated IL-1b, IL-6, and IL-
23 in the initiation and expansion of IL-17–producing T cells,
three cytokines that are highly induced by SAA (14, 41).
In this study, we report that multiple models of respiratory
system exposure that promote mixed Th2/Th17 responses also
induce pulmonary Saa3 expression. SAA signals through TLR2 to
induce inflammatory mediators and through the Nlrp3 inflamma-
some to induce IL-1b secretion. In addition, SAA induces den-
dritic cells (DCs) to undergo maturation and produce soluble
mediators, including IL-1a, IL-1b, IL-6, PGE
, and IL-23, that
function in an IL-1–dependent manner to promote CD4
T cells
to secrete IL-17A upon stimulation. Finally, SAA sensitizes mice
to a mixed Th2/Th17 allergic airway disease via an IL-1R–de-
pendent mechanism. Together, these data implicate pulmonary
SAA as a proinflammatory mediator capable of promoting Ag-
specific pulmonary Th17 responses through the activities of the
cytokine mediator IL-1.
Materials and Methods
C57BL/6 and IL-1Ra
mice were purchased from The Jackson Labo-
ratory (Bar Harbor, ME). TLR2
(42), TLR4
(43), MyD88
(45), ASC
(45), caspase-1
(46), and CC10-rtTA 3
TetOP-CAIKKb bitransgenic mice on the C57BL/6 background (47),
which express a constitutively active IkB kinase b (CAIKKb) in bron-
chiolar epithelium following administration of 6 g/kg doxycycline (Dox) in
chow (TestDiet, Richmond, IN), and age- and sex-matched transgene-
negative littermates were also bred at the University of Vermont. Mice
were housed in an American Association for the Accreditation of Labo-
ratory Animal Care-approved facility, maintained on a 12-h light/dark
cycle, and were provided food and water ad libitum. All animal studies
were approved by the University of Vermont Institutional Animal Care and
Use Committee.
For acute studies, mice were anesthetized with inhaled isoflurane and re-
ceived either 50 ml sterile saline, 100 ng ultra-pure LPS (InvivoGen, San
Diego, CA), or 10 mg apo-SAA (PeproTech, Rocky Hill, NJ) in 50 ml
sterile saline by oropharyngeal aspiration (o.a.), and analyzed 24 h later.
Mice were exposed to 15 ppm NO
or high-efficiency particulate air-fil-
tered room air for 1 h and analyzed 24 h later as previously described (48).
CAIKKb mice and transgene-negative littermates were provided Dox-
containing chow for 60 h prior to analysis. For treatment with anakinra
(Biovitrum, Stockholm, Sweden), mice were administered 1 mg drug in
200 ml sterile saline by s.c. injection twice daily beginning 1 d before SAA
exposure. For Ag-sensitization studies, mice received either saline or SAA
(as above) once on day 0, followed by 30 min of nebulized 1% OVA,
Fraction V (Sigma-Aldrich, St. Louis, MO), in saline on days 0, 1, and 2.
Mice were then challenged with 30 min of nebulized 1% OVA on days 14,
15, and 16 and analyzed on day 18. Alum/OVA-treated mice were sensi-
tized on day 0 with 100 mg OVA in Imject Alum (Thermo Scientific,
Rockford, IL), challenged with 30 min of nebulized 1% OVA on days 14,
15, and 16, and analyzed on day 18.
Bronchoalveolar lavage collection and lung processing
Lungs were lavaged with 1 ml DPBS (Sigma-Aldrich) from which cells
were counted by hemocytometer, and differential analysis was performed
by cytospin and H&E stain. After lavage, lungs were flash frozen in liquid
nitrogen for RNA analysis.
For the isolation of primary macrophages, C57BL/6, TLR2
, TLR4
and MyD88
mice were administered 1 ml 4% thioglycollate by i.p.
injection. Ninety-six hours later, mice were euthanized, and peritoneal
lavage was performed to collect peritoneal exudate cells. Cells were plated
in RPMI 1640 supplemented with 10% FBS, penicillin and streptomycin,
L-glutamine, and 2-ME and challenged for 16 h with apo-SAA followed
where indicated by 30 min of 5 mM ATP or 8 h of 500 mg/ml Imject Alum
(Thermo Scientific). C57BL/6, NLRP3
, and caspase-1
transformed macrophage cell lines (49) were maintained in vitro in RPMI
1640 with 10% FBS, penicillin and streptomycin,
L-glutamine, and 2-ME.
Following stimulation, cell-free supernatants were flash frozen for later
Cytokine analysis
Cytokines from bronchoalveolar lavage (BAL) and cell supernatants were
analyzed by ELISA for IL-1b and TNF-a (BD Biosciences, San Jose, CA),
as well as IL-23 (R&D Systems, Minneapolis, MN). PGE
was assessed by
enzyme immunoassay (Cayman Chemical, Ann Arbor, MI). Customized
Bio-Plex (Bio-Rad, Hercules, CA) and Milliplex assays (Millipore, Bill-
erica, MA) were used to measure IL-5, IL-13, and IL-17, as well as IL-1a,
IL-1b, TNF-a, IL-6, GM-CSF, G-CSF, keratinocyte-derived chemokine,
MIP-1a, MIP-1b, MCP-1, IL-12p40, and IL-12p70.
Quantitative RT-PCR
Total RNA was extracted from frozen whole lungs or transformed mac-
rophages using the PrepEase RNA Isolation kit (USB, Cleveland, OH) and
reversed transcribed to cDNA using the iScript kit from Bio-Rad. Primers
were designed for mouse Saa1, Saa2, Saa3, and Il1b, and RT-PCR was
performed using SYBR Green Supermix (Bio-Rad) and normalized to
Gapdh or Actb using the DDC
method, as previously described (47).
Splenocyte restimulation
Splenocytes from experimental mice and C57BL/6 control mice were
isolated using Lymphocyte Separation Media (MP Biomedicals, Solon, OH)
as previously described (48). A total of 4 3 10
cells/ml were cultured in
RPMI 1640 supplemented with 10% FBS, penicillin/streptomycin,
tamine, and 2-ME and were activated with 100 mg/ml OVA in 48-well
plates. Following 96 h of stimulation, supernatants were collected for
analysis by Milliplex (Millipore).
Bone marrow-derived DCs
Bone marrow was flushed from the femurs and tibiae and cultured on 24-
well plates at 1 3 10
cells/well (1 ml/well) in RPMI 1640 containing 10%
serum and 10% conditioned media from X63-GMCSF myeloma cells
transfected with murine GM-CSF cDNA (kindly provided by Dr. Brent
Berwin, Dartmouth College). Media was replaced on days 2 and 4, and the
adherent and lightly adherent bone marrow-derived DCs (BMDCs), pre-
dominantly CD11b
by FACS, were collected on day 6. BMDCs
were treated with SAA for 16 h. For flow cytometry, BMDCs were de-
tached using versene and gentle scraping, washed in FACS buffer (DPBS
with 5% FBS and 0.1% sodium azide), and 1 3 10
cells were incubated
with Fc block (2.5 mg/ml anti-CD16/CD32) (BD Pharmingen) for 30 min
at 4˚C, washed in FACS buffer, and then stained for 30 min at 4˚C in 100
ml Ab solution at the optimal concentration. Cells were stained with: anti-
CD80–PE (BD Pharmingen), anti-CD86–Alexa 647 (Caltag Laboratories,
Carlsbad, CA), anti-MHC class II-PerCP/Cy5.5 (BD Pharmingen), and
biotinylated anti-OX40L (BD Pharmingen). Biotinylated Abs were
detected using streptavidin-PE (BD Pharmingen). Following staining, all
The Journal of Immunology 65
Page 2
cells were washed and fixed in DPBS with 5% FBS and 1% para-
formaldehyde. Cells were analyzed on an LSR II FACS flow cytometer
(BD Biosciences) equipped to distinguish as many as seven fluorophores
1–3 d following staining. Dead cells were excluded from analysis by
forward light scatter and side scatter gating. Data were analyzed using
FlowJo (Tree Star, Ashland, OR).
SAA contaminant analysis
BMDCs were treated with SAA for 16 h in the presence or absence of
polymyxin B (Sigma-Aldrich) at 25 and 1 mg/ml. Proteinase K (Sigma-
Aldrich) at 25 mg/ml and 1 mg/ml (or absent) was incubated with apo-SAA
at 37˚C for 1 h, heated to 100˚C for 5 min to deactivate the enzyme, and
allowed to cool to room temperature before addition to cells. Cells were
treated for 16 h, and cell-free supernatants were flash frozen prior to
further analysis.
T cell culture with BMDC-conditioned media
Cell-free conditioned media from unstimulated or 24 h SAA-exposed
BMDCs were incubated with 1 3 10
splenic CD4
T cells from naive
mice that were stimulated in the presence of 5 mg/ml immobilized anti-
CD3 and 1 mg/ml soluble anti-CD28 for 96 h. Alternatively, CD4
T cells
were polyclonally stimulated in media alone or in the presence of 1 mg/ml
Splenocyte cultures
Splenocytes from C57BL/6, TLR2
, and caspase-
mice were cultured at 4 3 10
cells/ml in RPMI 1640 containing
10% FBS, penicillin/streptomycin,
L-glutamine, and 2-ME on plates
coated with 5 mg/ml anti-CD3 and treated with 1 mg/ml soluble anti-CD28,
10 ng/ml anakinra, and 1 mg/ml SAA. Cell-free supernatants were ana-
lyzed 96 h after stimulation.
Data were analyzed by two-tailed unpaired t test or one-way ANOVA and
Bonferroni post hoc test using GraphPad Prism 4 for Windows (Graph-
Pad). A p value ,0.05 was considered statistically significant.
SAA3 expression in mouse lung
Our studies have employed multiple mechanisms of allergic sen-
sitization, including NO
exposure, oropharyngeal administration
of LPS, and airway epithelial-specific NF-kB activation, each of
which can promote mixed Th2/Th17 responses (40, 47, 48). Fol-
lowing exposure to these stimuli, lungs of C57BL/6 mice
exhibited a preferential mRNA induction of Saa3 over Saa1 or
Saa2 (Fig. 1A). Mice exposed to 15 ppm NO
for 1 h and analyzed
24 h later showed a 6-fold induction of Saa3 in the lung, very
similar to the response in transgenic mice that inducibly express
CAIKKb in the airway epithelium following 48 h of Dox ad-
ministration (Fig. 1A). Oropharyngeal administration of 100 ng
LPS, a low dose used in models of inhalational allergic sensiti-
zation (40, 50), induced high mRNA levels of Saa3 24 h post-
challenge (Fig. 1A). These results demonstrate expression of Saa3
in the lung under conditions that facilitate mixed Th2/Th17 po-
FIGURE 1. SAA is expressed in the lungs during
mixed Th2/Th17 allergic sensitization regimens and
induces pulmonary inflammation upon inhalational
exposure. Quantitative PCR of whole lung for SAA
isoforms in mice exposed to NO
, LPS, or in which
NF-kB has been activated in the airway epithelium
(A). C57BL/6 mice were administered 10 mg SAA
by o.a. and analyzed 4 and 24 h later. BAL total cell
counts were performed by hemocytometer, and dif-
ferential analysis was by cytospin (B). BAL fluid
was analyzed by Milliplex assay (Millipore) for
IL-1b (C), TNF-a (D), IL-6 (E), GM-CSF (F),
G-CSF (G), keratinocyte-derived chemokine (H),
MIP-1a (I), MIP-1b (J), MCP-1 (K), IL-12p40 (L),
and IL-12p70 (M). Data are representative of three
independent experiments. C57BL/6 mice were ad-
ministered saline or 1 mg anakinra (n = 3 per group)
by s.c. injection twice daily beginning one day prior
to o.a. of 10 mg SAA. At 24 h, BAL total cell counts
were performed by hemocytometer and differential
analysis was by cytospin (N). *p , 0.05, **p ,
0.005, ***p , 0.001 compared with control expo-
sures (A) or saline controls (BN).
Page 3
SAA elicits robust IL-1–dependent pulmonary inflammation
Recombinant human apo-SAA (with functional similarity to mouse
SAA3) is available commercially from PeproTech and reported to
contain ,1 EU/mg endotoxin. Our own Limulus amebocyte lysate
assay confirmed that this was indeed true (data not shown). To
determine the effects of SAA in the lung, mice were administered
10 mg apo-SAA or saline by o.a. and analyzed at 4 and 24 h. In
contrast to saline, SAA induced robust airway neutrophilia (Fig.
1B) and the production of inflammatory cytokines, as measured
from BAL fluid (Fig. 1CM). Because IL-1b was present at ele-
vated concentrations in the BAL fluid (Fig. 1C), we administered
an IL-1R antagonist (anakinra) or saline to mice prior to apo-SAA
aspiration. In the anakinra-treated mice, airway neutrophilia was
significantly reduced compared with apo-SAA–exposed mice
treated with saline vehicle (Fig. 1N). These results implicate an
important function of IL-1 in SAA-promoted inflammation.
SAA activates DCs that promote IL-17A production from CD4
T cells
To determine the effects of SAA on APCs, which are critical for
initiation of CD4
T cell responses, BMDCs from C57BL/6 mice
were challenged with apo-SAA and analyzed 16 h later. We
FIGURE 2. SAA elicits inflammatory medi-
ator production and DC maturation in vitro.
BMDCs were treated with 1 mg/ml SAA for 16
h and analyzed for surface markers of matu-
ration (A) and secretion of Th17-polarizing
mediators (B). The conditioned media from
control or SAA-exposed BMDCs (C) or fresh
media with or without SAA (D) was transferred
to CD4
T cells that were polyclonally stimu-
lated with anti-CD3 and anti-CD28. After 96 h,
IFN-g, IL-4, and IL-17A were measured in
supernatants. Data are representative of three
independent experiments. *p , 0.05, ***p ,
0.001 compared with control exposures.
FIGURE 3. SAA-induced IL-1b production
requires TLR2, MyD88, and the NLRP3 inflam-
masome. Cell-free supernatants from BMDCs
treated with SAA or LPS in the presence or ab-
sence of proteinase K (A) or polymyxin B (B)
were analyzed by ELISA after 24 h. Peritoneal
exudate macrophages from wild-type (C57BL/6),
, and MyD88
mice were
unstimulated or primed overnight with 1 mg/ml
SAA alone or followed by 30 min of 5 mM ATP or
8 h of 500 mg/ml Alum, and supernatants were
analyzed for IL-1b secretion (C). Transformed
macrophages from wild-type (C57BL/6), Nlrp3
, and caspase-1
mice were treated for
24 h with 1 mg/ml SAA, and supernatants were
analyzed for TNF-a (D) and IL-1b (E) secretion.
Il1b expression was measured from wild-type
(C57BL/6), Nlrp3
, and caspase-1
transformed macrophages that had been untreated
orexposedfor4hto1mg/ml SAA (F). **p ,
0.005, ***p , 0.001 compared with wild-type (C)
or control exposures (DF).
The Journal of Immunology 67
Page 4
observed increases in surface markers of DC maturation, including
CD80, CD86, MHC class II, and OX40L (Fig. 2A), upon exposure
of BMDCs to apo-SAA. In addition, BMDCs secreted IL-1a, IL-
1b, IL-6, and IL-23 (Fig. 2B), cytokines that participate in Th17
polarization and maintenance (10, 23, 51). Furthermore, treatment
with SAA caused BMDCs to secrete a significant amount of
(Fig. 2B), which has recently been shown to induce DCs
to preferentially secrete IL-23 and thus contribute to the de-
velopment of a Th17 response (52, 53). DCs exposed to apo-SAA
also secreted small amounts of the Th1-polarizing cytokine, IL-
12p70, and substantial amounts of proinflammatory TNF-a (Fig.
2B). When the cell-free supernatants from these SAA-treated
BMDCs were provided to polyclonally stimulated naive CD4
T cells, a significant production of IL-17 was induced compared
with very small amounts of IFN-g and IL-4 (Fig. 2C). Importantly,
treatment of the polyclonally stimulated naive CD4
T cells with
SAA resulted in no significant production of any of these cyto-
kines (Fig. 2D), indicating that the effect of SAA is directly on
DCs, which in turn drive the polarization of the CD4
T cells
through the secretion of soluble mediators, including IL-1b.
SAA-induced IL-1b secretion requires TLR2 and the NLRP3
Because IL-1b has been implicated as a Th17-polarizing and
-priming factor, we determined cell receptors required for SAA-
induced secretion of this cytokine. To ensure that the effects of
SAA required the recombinant SAA protein and were not due to
endotoxin contamination, we cultured BMDCs for 16 h with
apo-SAA or LPS that had been treated with proteinase K or left
untreated. All samples were then boiled to inactivate the pro-
teinase K. Under these conditions, we observed a dose-dependent
reduction in the response to apo-SAA, but no effect on the LPS-
induced IL-1b response (Fig. 3A). In contrast, exposure of
BMDCs to LPS in the presence of polymyxin B completely
blocked IL-1b production, whereas the presence of polymyxin B
during apo-SAA treatment still allowed for substantial amounts of
IL-1b to be produced (Fig. 3B). Having demonstrated the re-
quirement for SAA protein and the minimal contribution of con-
taminating endotoxin in the effects of apo-SAA, we exposed
peritoneal exudate macrophages from TLR2
, TLR4
, and
mice to SAA for 16 h. In these cells, IL-1b secretion
is primarily dependent upon TLR2 and MyD88, both in the ab-
sence or presence of ATP or aluminum crystals (Fig. 3C), potent
inducers of IL-1b secretion. TLR4
peritoneal exudate cells
showed no significant reduction in their ability to secrete IL-1b
following SAA treatment, further ruling out the contribution of
endotoxin contamination of apo-SAA to its biological effects in
these experimental systems. The response to SAA was also ex-
amined in transformed macrophage cell lines from wild-type,
, and caspase-1
mice. Whereas apo-
SAA–induced levels of TNF-a, an inflammasome-independent
cytokine, were highly induced in all cells (Fig. 3D), complete
abrogation of IL-1b secretion was observed in response to apo-
SAA in NLRP3-, ASC-, and caspase-1–deficient macrophages
(Fig. 3E). Indicative of the roles these molecules play in pro-
cessing the pro–IL-1b protein, levels of Il1b message expression
were robustly induced in the wild-type, NLRP3-, ASC-, and
caspase-1–deficient macrophages (Fig. 3F).
SAA-induced pulmonary inflammation requires TLR2 and
involves the NLRP3 inflammasome
Having demonstrated the important functions of TLR2, NLRP3,
and caspase-1 for SAA-induced IL-1 b secretion in vitro, we next
performed additional pulmonary SAA exposures to determine the
contribution of these molecules to inflammation in vivo. Oro-
pharyngeal administration of 10 mg apo-SAA elicited a robust
influx of neutrophils into the lung after 24 h in C57BL/6 mice,
a response that was diminished in TLR2
, but not NLRP3
FIGURE 4. SAA-induced pulmonary inflamma-
tion requires TLR2 and involv es the NLRP3
inflammasome. Wild-type (C57BL/6), TLR2
, and caspase-1
mice were administer ed
10 mgSAAbyo.a.andanalyzed24hlater.BAL
total cell counts were performed by hemocyto-
meter and differential analysis was by cytospin (A).
Il1b expression was measured from whole lung by
quantitative R T-PCR (B). BAL fluid was ana-
lyzed by Milliplex assay (Millipore) for IL-1b (C),
G-CSF (D), IL-6 (E), and MCP-1 (F). Data are re-
presentative of three independent experiments. *p ,
0.05, **p , 0.005, ***p , 0.001 compared with
wild-type (A)orsaline(BF).
Page 5
, mice (Fig. 4A). Measurement of Il1b gene expres-
sion in the lung following SAA aspiration revealed increased
mRNA abundance in wild-type, NLRP3
, and caspase-1
mice, but not in TLR2
mice (Fig. 4B). Analysis of the BAL
fluid from these mice demonstrated that TLR2 signaling was also
required for IL-1b, G-CSF, IL-6, and MCP-1 production, whereas
NLRP3 and caspase-1 were necessary only for the production of
IL-1b (Fig. 4C–F). The NLRP3
and caspase-1
mice also
displayed reduced levels of IL-6 and MCP-1, two cytokines that
can be induced by IL-1 b (54, 55) and are perhaps diminished as
a consequence of lost IL-1b signaling.
SAA-promoted allergic sensitization favors a Th17 response
that requires IL-1Ra signaling
Having demonstrated evidence for DC maturation and Th17
polarization in response to apo-SAA in vitro, we sought to com-
pare a conventional Th2 allergic sensitization protocol, a well-
characterized Alum/OVA model (56, 57), with an experimental
model of apo-SAA–promoted Ag sensitization (Fig. 5A). A very
distinct profile of SAA gene expression occurred in the lung 24 h
following Ag sensitization in the two models (Fig. 5B). In-
traperitoneal injection of the adjuvant Alum elicited little SAA
expression in the lung (Fig. 5B; 3.4-fold induction of Saa1, 6.9-
fold induction of Saa2, and 3.0-fold induction of Saa3), but in-
duced strong expression of Saa1 (781-fold induction) and Saa2
(199-fold induction) in the liver, with only 4.2-fold induction of
Saa3 (data not shown). This is consistent with the reported roles of
liver SAA1 and SAA2 in the systemic inflammatory response. In
contrast, pulmonary administration of apo-SAA by o.a. selectively
induced mRNA expression of Saa3 in the lungs (Fig. 5B), with no
systemic (liver) production of any other isotypes (data not shown).
The disparate induction of Saa expression in liver and lung led
us to speculate that differences in route of sensitization and ad-
juvant used modulate local effects that may contribute to distinct
responses in the Alum- and SAA-promoted allergic sensitization
models. Therefore, we examined the BAL cell profiles from
challenged mice subjected to the two models of Ag sensitization.
Mice sensitized i.p. with Alum/OVA robustly recruited eosino-
phils into the lung compared with unsensitized saline/OVA con-
trols, whereas the SAA/OVA sensitized mice showed eosinophilia
on the order of 10–20%, which is more representative of that
typically present in the BAL fluid in an asthmatic patient (Fig.
5C). Splenocytes from sensitized and control mice were cultured
and restimulated with OVA for 96 h. The Alum/OVA mice, as
expected, responded by producing copious amounts of the Th2
cytokines IL-5 and IL-13, whereas SAA/OVA mice produced
modest but elevated levels of these cytokines compared with the
saline control mice (Fig. 5D,5E). However, SAA/OVA-sensitized
FIGURE 5. SAA inhalation promotes Th17 allergic sensitization. Mice underwent Ag sensitization via o.a. with either saline and OVA (saline/OVA) or
SAA and OVA (SAA/OVA) or via i.p. injection with either saline and OVA (saline/OVA) or Alum and OVA (Alum/OVA), according to the schema (A).
Saa1, Saa2, and Saa3 gene expression in whole lung was measured on day 1, 24 h after i.p. injection with saline or Alum or 24 h after o.a. administration of
saline or SAA (B). On day 18, total cell counts from BAL fluid were performed by hemocytometer, and differential analysis was by cytospin (C). On day
18, splenocytes from i.p. saline, i.p. Alum, o.a. saline, and o.a. SAA mice were restimulated in vitro with OVA for 96 h, and IL-5 (D), IL-13 (E), and IL-17A
(F) levels in culture media were measured. Data are representative of three independent experiments. *p , 0.05, **p , 0.005, ***p , 0.001 compared with
saline controls (B, DF).
The Journal of Immunology 69
Page 6
mice, unlike Alum/OVA mice, displayed significant production of
IL-17 in response to Ag (Fig. 5F), recapitulating our in vitro
findings from Fig. 2C that the SAA-induced inflammatory re-
sponse can polarize T cells to secrete primarily IL-17A.
As we have demonstrated, the capacity of Ag-restimulated
splenocytes to produce IL-17 may rely on the microenvironmen-
tal cytokine milieu that is generated by DCs and other APCs in
response to SAA. Because SAA promotes IL-1–dependent pul-
monary inflammation, we repeated our SAA/OVA sensitization
model (Fig. 5A) using IL-1Ra knockout mice to determine
whether IL-1 signaling played a critical role in the CD4
T cell
priming and polarization process. Following Ag challenge, the
cellular BAL profile revealed that IL-1Ra
mice exposed to
SAA/OVA recruited fewer eosinophils and lymphocytes to the
lung than did wild-type mice (Fig. 6A). Furthermore, when
splenocytes from these sensitized and challenged mice were
restimulated in vitro with OVA, wild-type and knockouts showed
a similar induction of IL-5 and IL-13, but the IL-17A production
was absent in the IL-1Ra
mice (Fig. 6B–D). To show that an
IL-1Ra ligand was required for SAA to induce IL-17A production
and that the IL-17A deficiency seen in IL-1Ra
mice was not
due to a developmental defect, splenocytes from wild-type mice
were polyclonally stimulated in the presence of SAA, with or
without anakinra. The addition of anakinra, even at a relatively
low dose of 10 ng/ml, completely abrogated IL-17A production
(Fig. 6E). Finally, to investigate the requirement for TLR2 and
NLRP3 inflammasome components for SAA-promoted IL-17A
production, splenocytes from C57BL/6, TLR2
, and caspase-1
mice were plated and polyclonally
stimulated in the presence or absence of apo-SAA. The TLR2
splenocytes showed a nearly complete inability of SAA to aug-
ment IL-17A production after 96 h of stimulation (Fig. 6F). IL-17
production was also impaired in splenocytes that lacked compo-
nents of the NLRP3 inflammasome complex, although the abro-
gation was more moderate (Fig. 6F). Clearly, IL-1 signaling plays
a critical role in the induction of SAA-promoted allergic airway
disease, specifically in mediating the production of IL-17A.
Our results reported in this study implicate a causal role and
a molecular mechanism for SAA in the pathogenesis of allergic
asthma. Several of our previous studies have focused on in-
halational Ag sensitization via exogenous insults, predominantly
the pollutant NO
. Inhalation of NO
activates airway epithelial
NF-kB and promotes a mixed Th2/Th17 response (48, 58), which
can be recapitulated through the activation of NF-kB in airway
epithelium and inhalation of Ag (47). Within the lung, SAA3 is
induced as a consequence of both NO
inhalation and airway
epithelial NF-kB activation. SAA isotypes are differentially ex-
pressed in distinct tissues (13). SAA1 and - 2 in mice are
expressed predominantly in the liver, most commonly found
bound to high-density lipoproteins in the circulation, and acutely
upregulated in systemic disease states (13, 59). In contrast, SAA3
in mice has been shown to be expressed in a wide variety of cells
and tissues, including leukocytes and epithelium, and has never
been identified bound to high-density lipoproteins (60–62). In
humans, SAA1 and -2 are expressed in the liver and in the lung
and have been associated locally with the TLR2-dependent de-
velopment of sarcoidosis (63). The rapid and robust induction of
SAA in response to a panel of inhalational stimuli (Fig. 1A)
indicates a possible role for SAA as a mediator in both allergic
sensitization and during Ag challenge (exacerbation). Based on
our results, we speculate that local production of SAA, rather than
the particular isoform expressed, is capable of influencing local
FIGURE 6. SAA-promoted allergic
sensitization and Th17 polarization re-
quire IL-1R. C57BL/6 and IL-1Ra
mice were Ag-sensitized with saline and
OVA (saline) or SAA and OVA (SAA) by
o.a., according to the timeline in Fig. 5A.
On day 18, total and differential cell
counts from BAL fluid were performed
(A). Splenocytes were restimulated in
vitro with OVA for 96 h, and IL-5 (B),
IL-13 (C), and IL-17 (D) levels in culture
media were measured. Splenocytes from
C57BL/6 mice in the presence or absence
of SAA and 10 ng/ml anakinra (E) and
splenocytes from C57BL/6, TLR2
, and caspase-1
(F) mice were polyclonally stimulated for
96 h with anti-CD3 and anti-CD28 in the
presence or absence of 1 mg/ml SAA, and
IL-17A was measured by ELISA (F). Data
are representative of two independent
experiments. *p , 0.05, **p , 0.005,
***p , 0.001 compared with saline con-
trols (AD), SAA (E), or wild-type (F).
Page 7
innate and adaptive immune responses. Therefore, whereas SAA3
is the predominant form of SAA expressed in mouse lungs, SAA1
or SAA2 in human lungs may exert effects similar to those we
report in this study.
The inflammatory cytokine milieu surrounding naive DCs is key
to their maturation and to the polarization of the CD4
T cell
response, as we have previously demonstrated using a model of
inducible airway epithelial NF-kB activation (47). In addition, the
work of Ivanov et al. (20) has implicated SAA as an important
mediator for Th17 polarization in the gut in response to coloni-
zation with segmented filamentous bacteria. We have shown
a profound effect of apo-SAA on BMDCs that includes APC
maturation and the secretion of the Th17-polarizing mediators
IL-1a, IL-1b, IL-6, IL-23, and PGE
. From the studies presented
in this article using IL-1R antagonism with anakinra, it is clear
that IL-1a and IL-1b are potential mediators induced by SAA that
are capable of eliciting predominantly IL-17A production from
naive, polyclonally stimulated CD4
T cells.
IL-1b requires cleavage via caspase-1 for proper secretion,
which is facilitated as a consequence of inflammasome assembly
and activation. The NLRP3 inflammasome has emerged as a crit-
ical cytosolic sensor for a number of endogenous mediators, in-
cluding amyloid proteins (2, 22), that are capable of promoting
IL-1b secretion. Our studies demonstrate that regulation of IL-1 b
production in response to SAA occurs as two distinct levels. At the
transcriptional level, SAA-induced IL-1b requires TLR2, a finding
first reported in human ThP-1 cells by Cheng et al. (14). At the
posttranslational level, use of transgenic mice that are deficient
in proteins of the NLRP3 complex demonstrate a clear role for
the NLRP3 inflammasome in SAA-induced IL-1b secretion.
mice exhibit severe impairment in the inflammatory
response to oropharyngeal administration of apo-SAA, including
a diminution in neutrophil recruitment and decreased secretion of
inflammatory cytokines in the BAL. The remaining neutrophilia in
these mice is likely due to the capacity of apo-SAA to stimulate
neutrophil chemotaxis via FPRL1/FPR2 (17, 18), which remains
intact in all of the mice we studied. Taken together, our in vitro
and in vivo results indicate that proper production and secretion of
IL-1b in response to SAA is regulated both at the transcriptional
level by TLR2 and at the level of secretion by the NLRP3
inflammasome. Furthermore, they suggest that IL-1b not only
participates in the inflammatory cascade, but also amplifies the
response through the augmented production of select inflam-
matory cytokines. Nevertheless, our in vivo data using anakinra,
an antagonist of the IL-1R that blocks the effects of both IL-1a
and IL-1b, demonstrate a causal role for IL-1 in SAA-pro-
moted pulmonary neutrophilia, whereas the in vivo data from
the NLRP3- and caspase-1–deficient mice reveal very modest
reductions in inflammatory cytokines (aside from IL-1b) and
pulmonary neutrophilia following SAA aspiration. Taken together,
these findings suggest that there may be an additional role for IL-
1a release in response to SAA that requires further investigation.
Models of in vitro and in vivo Th17 polarization require the
presence of IL-1, IL-23, and IL-6 (23, 64–67) and repression of
Th1- and Th2-polarizing cytokines to establish a unique envi-
ronment that challenge with apo-SAA appears to replicate. The
Th2-polarizing Alum/OVA model has long been criticized for the
magnitude of the response, whereas inhalational models of aller-
gic sensitization (using different adjuvants such as LPS or ciga-
rette smoke) tend to induce a modest eosinophilia that is more
representative of human asthma (68). The more moderate response
seen in our SAA/OVA sensitization model could represent a dif-
ferent aspect of the asthma syndrome, one that is less Th2 in
nature and more Th17. It has recently been shown that IL-17
production promoted by IL-1b involves conversion of Foxp3
T regulatory cells to retinoic acid-related orphan receptor gt-
expressing Th17 cells (10, 11). In addition, in the human disease
and in mouse models of severe allergic asthma, CD4
T cell
production of IL-17 can originate from a population of CRTh2
effector cells/memory cells that are capable of secreting Th2
cytokines and coexpressing the transcription factors GATA3 and
retinoic acid-related orphan receptor gt, a population that can be
induced upon stimulation with proinflammatory cytokines, in-
cluding IL-1b (69). Our studies have not identified the precursor
T cell population that develops the capacity to produce
IL-17A in response to SAA, nor have we yet thoroughly in-
vestigated the spectrum of Th17-related cytokines produced by
these IL-17A–producing CD4
T cells. Regardless of the mech-
anism of CD4
T cell conversion, the role that SAA may play in
the process as an endogenous mediator has far-reaching implica-
tions for the pathogenesis of IL-17–producing CD4
T cells in
severe allergic asthma.
The conclusions of the studies described in this study are 3-fold.
First, it is clear that the acute-phase SAA proteins are more than
simply biomarkers of disease severity. Instead, they function as
biological mediators through the stimulation of TLR2 and the
NLRP3 inflammasome to regulate pulmonary cytokine production
and neutrophilia. Second, although the properties of SAA that
enable activation of these pathways remain to be determined, the
capacity for both to induce IL-1b gene expression and allow for
IL-1b secretion make SAA distinct from other endogenous amy-
loid peptides and proteins that function solely in the later step of
IL-1b release (2, 22). Third, SAA is sufficient to function as an
adjuvant to promote allergy to an innocuous inhaled Ag in
a manner that is dependent upon IL-1R signaling to stimulate the
capacity of CD4
T cells to produce IL-17A. Whether SAA is
necessary for Th17 development in response to inhalational Ag
sensitization and at what threshold concentration endogenous
SAA manifests TLR2/NLRP3 stimulation remain to be de-
termined. These results are the first, to our knowledge, to link
pulmonary SAA, IL-1b, and IL-17A in a manner that explains
their interrelationships in allergic asthma and their mechanisms of
action. Based upon our findings, it is evident that novel models of
SAA/IL-1–mediated allergic airway disease may provide new
insight into the endogenous mechanisms behind the inappropriate
or maladaptive immune responses at play in the complex pheno-
types of allergic asthma.
The authors have no financial conflicts of interest.
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    • "This data suggested that the early increase in mitochondrial-derived ROS is directly related to synthesis of intracellular pro-IL1β but not to the secretion of IL-1β in CNC-AEMA2 stimulated cells. Several studies reinforce the idea that mitochondrial ROS, especially generated as a consequence of mitochondrial dysfunction, are the main source of ROS participating in NLRP3 inflammasome activation202122232425262728293031. Although, it appears that CNC-AEMA2 action is mainly through mitochondrion, this compound was not the only one to cause mitochondrial alterations. "
    [Show abstract] [Hide abstract] ABSTRACT: Crystalline cellulose nanocrystals (CNCs) have emerged as novel materials for a wide variety of important applications such as nanofillers, nanocomposites, surface coatings, regenerative medicine and potential drug delivery. CNCs have a needle-like structure with sizes in the range of 100-200. nm long and 5-20. nm wide and a mean aspect ratio 10-100. Despite the great potential applicability of CNCs, very little is known about their potential immunogenicity. Needle-like materials have been known to evoke an immune response in particular to activate the (NOD-. like receptor, pyrin domain-containing 3)-inflammasome/IL-1β (Interleukin 1β) pathway. In this study we evaluated the capacity of unmodified CNC and its cationic derivatives CNC-AEM (aminoethylmethacrylate)1, CNC-AEM2, CNC-AEMA(aminoethylmethacrylamide)1 and CNC-AEMA2 to stimulate NLRP3-inflammasome/IL-1β pathway and enhance the production of mitochondrial reactive oxygen species (ROS). Mouse macrophage cell line (J774A.1) was stimulated for 24. h with 50. μg/mL with unmodified CNC and its cationic derivatives. Alternatively, J774A1 or PBMCs (peripheral blood mononuclear cells) were stimulated with CNC-AEMA2 in presence or absence of LPS (lipopolysaccharide). IL-1β secretion was analyzed by ELISA, mitochondrial function by JC-1 staining and ATP content. Intracellular and mitochondrial reactive oxygen species (ROS) were assessed by DCF-DA (2',7'-dichlorodihydrofluorescein diacetate) and MitoSOX, respectively. Mitochondrial ROS and extracellular ATP were significantly increased in cells treated with CNC-AEMA2, which correlates with the strongest effects on IL-1β secretion in non-primed cells. CNC-AEMA2 also induced IL-1βsecretion in LPS-primed and non-primed PBMCs. Our data suggest that the increases in mitochondrial ROS and ATP release induced by CNC-AEMA2 may be associated with its capability to evoke immune response. We demonstrate the first evidence that newly synthesized cationic cellulose nanocrystal derivative, CNC-AEMA2, has immunogenic properties, which may lead to the development of a potential non-toxic and safe nanomaterial to be used as a novel adjuvant for vaccines.
    Full-text · Article · Dec 2015
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    • "SAA also possesses proinflammatory properties that induce the release of cytokines from different cell types, including monocytes [1], [2]. Recent studies showed that SAA induced the expression of pro-IL-1β and activated the NRLP3 inflammasome, resulting in the secretion of mature IL-1β [3]–[5]. IL-1β is a key proinflammatory cytokine with a central role in the damaging inflammatory processes that accompany sterile disease [6]. "
    [Show abstract] [Hide abstract] ABSTRACT: Background/AimsSerum amyloid A (SAA) is an acute phase reactant with significant immunological activities, including effects on cytokine synthesis and neutrophil chemotaxis. Neutrophils can also release cytokines with proinflammatory properties. IL-1β is a key proinflammatory cytokine, the secretion of which is controlled by inflammasome. We investigated the proinflammatory effects of SAA in vitro in relation to the NLRP3 inflammasome in neutrophils.Methodology/Principal FindingsHuman neutrophils isolated form healthy subjects were stimulated with serum amyloid A (SAA). The cellular supernatants were analyzed by western blot using anti-IL-1β or anti-caspase-1 antibodies. IL-1β or Nod-like receptor family, pyrin domain containing 3 (NLRP3) mRNA expressions were analyzed by real-time PCR or reverse transcription-PCR (RT-PCR) method. SAA stimulation induced pro-IL-1β mRNA expression in neutrophils. Furthermore, SAA engaged the caspase-1-activating inflammasome, resulting in the production of active IL-1β. SAA-induced pro-IL-1β expression was marginally suppressed by the Syk specific inhibitor, R406, and SAA-induced pro-IL-1β processing in neutrophils was prevented by R406. Furthermore, SAA-induced NLRP3 mRNA expression was completely blocked by R406. Analysis of intracellular signaling revealed that SAA stimulation activated the tyrosine kinase Syk and mitogen-activated protein kinase (MAPK).Conclusions/SignificanceThese results demonstrate that the innate neutrophil immune response against SAA involves a two-step activation process: an initial signal promoting expression of pro-IL-1β and a second signal involving Syk-dependent activation of the NLRP3 inflammasome and caspase-1, allowing processing of pro-IL-1β and secretion of mature IL-1β.
    Full-text · Article · May 2014 · PLoS ONE
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    • "pro-inflammatory cytokines (including IL-1b) and nitric oxide from neutrophils, macrophages and epithelial cells (Ather et al., 2011; Hiratsuka et al., 2008). SAA3 instillation into the lungs elicits robust pro-inflammatory cytokine production and phagocyte recruitment into the lungs (Ather et al., 2011 ). Our previous data show significant IL- 1b increases in the lungs of H99c-immunized mice that are protected against experimental pulmonary C. neoformans infection (Wozniak et al., 2009), which could be induced by mediators such as SAA3. "
    [Show abstract] [Hide abstract] ABSTRACT: Cryptococcus neoformans is a significant cause of fungal meningitis in patients with impaired T cell-mediated immunity (CMI). Experimental pulmonary infection with a C. neoformans strain engineered to produce interferon-gamma, H99γ, results in the induction of Th1-type CMI, resolution of the acute infection, and protection against challenge with wild type (WT) Cryptococcus. Considering that individuals with suppressed T CMI are highly susceptible to pulmonary C. neoformans infection, we sought to determine whether or not antimicrobial peptides were produced in mice inoculated with H99γ. Thus, we measured the levels of antimicrobial peptides Lipocalin-2, S100A8, S100A9, calprotectin (S100A8/A9 heterodimer), serum amyloid A-3 (SAA3), and their putative receptors TLR4 and the receptor for advanced glycation end products [RAGE] in mice during primary and recall responses against C. neoformans infection. Results showed increased levels of IL-17A and IL-22, cytokines known to modulate antimicrobial peptide production. We also observed increased levels of Lipocalin-2, S100A8, S100A9, and SAA3 as well as TLR4+ and RAGE+ macrophages and dendritic cells in mice inoculated with H99γ compared to WT H99. Similar results were observed in the lungs of H99γ-immunized, compared to heat-killed C. neoformans-immunized, mice following challenge with WT yeast. However, IL-22 deficient mice inoculated with H99γ demonstrated antimicrobial peptide production and no change in survival rates compared to WT mice. These studies demonstrate that protection against cryptococcosis is associated with increased production of antimicrobial peptides in the lungs of protected mice that are not solely in response to IL-17A and IL-22 production and may be coincidental rather than functional.
    Full-text · Article · Apr 2014 · Microbiology
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