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.

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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]. "
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    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.
    PLoS ONE 05/2014; 9(5):e96703. DOI:10.1371/journal.pone.0096703 · 3.23 Impact Factor
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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. "
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    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.
    Microbiology 04/2014; 160(Pt_7). DOI:10.1099/mic.0.073445-0 · 2.56 Impact Factor
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    • "SAAs are also involved in the recruitment and activation of leukocytes during the APR [11]. In particular, SAAs can induce the expression of pro-IL-1β through interaction with TLR2 and TLR4 which then activates the NLRP3 inflammasome [13], [14], [15]. "
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    ABSTRACT: Sepsis rapidly activates the host inflammatory response and acute phase response. Severe sepsis, complicated by multiple organ failure, is associated with overwhelming inflammation and high mortality. We previously observed that zinc (Zn) deficiency significantly increases mortality in a mouse model of polymicrobial sepsis due to over-activation of the inflammatory response. In order to identify potential mechanisms that account for Zn-responsive effects, we generated whole exome expression profiles from the lung tissue of septic mice that were maintained on Zn modified diets. Based on systems analysis, we observed that Zn deficiency enhances the acute phase response and particularly the JAK-STAT3 pathway, resulting in increased serum amyloid A production. In vitro studies of primary hepatocytes and HepG2 cells substantiated that Zn-deficiency augments serum amyloid A production through up-regulation of the JAK-STAT3 and NF-κB pathways. In contrast, Zn inhibited STAT3 activation through the up-regulation of SHP1 activity. Collectively, these findings demonstrate that Zn deficiency enhances the acute phase response through up-regulation of the JAK-STAT3 pathway, thereby perpetuating increased inflammation that may lead to increased morbidity and mortality in response to sepsis.
    PLoS ONE 04/2014; 9(4):e94934. DOI:10.1371/journal.pone.0094934 · 3.23 Impact Factor
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