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Cited by 369Inflammasome and immune disordersNeuroinflammation and Neurodegeneration MechanismsImmune Response and Inflammation

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NLRP3 Inflammasome Is Expressed and Functional in Mouse Brain Microglia but Not in Astrocytes

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Gustin, Audrey, Kirchmeyer, Mélanie, Koncina, Eric, Felten, Paul, Losciuto, Sophie, Heurtaux, Tony, Tardivel, Aubry, Heuschling, Paul, Dostert, Catherine·2015·PLoS ONE
DOI 10.1371/journal.pone.0130624PMC PMC4474809
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Abstract

Neuroinflammation is the local reaction of the brain to infection, trauma, toxic molecules or protein aggregates. The brain resident macrophages, microglia, are able to trigger an appropriate response involving secretion of cytokines and chemokines, resulting in the activation of astrocytes and recruitment of peripheral immune cells. IL-1β plays an important role in this response; yet its production and mode of action in the brain are not fully understood and its precise implication in neurodegenerative diseases needs further characterization. Our results indicate that the capacity to form a functional NLRP3 inflammasome and secretion of IL-1β is limited to the microglial compartment in the mouse brain. We were not able to observe IL-1β secretion from astrocytes, nor do they express all NLRP3 inflammasome components. Microglia were able to produce IL-1β in response to different classical inflammasome activators, such as ATP, Nigericin or Alum. Similarly, microglia secreted IL-18 and IL-1α, two other inflammasome-linked pro-inflammatory factors. Cell stimulation with α-synuclein, a neurodegenerative disease-related peptide, did not result in the release of active IL-1β by microglia, despite a weak pro-inflammatory effect. Amyloid-β peptides were able to activate the NLRP3 inflammasome in microglia and IL-1β secretion occurred in a P2X7 receptor-independent manner. Thus microglia-dependent inflammasome activation can play an important role in the brain and especially in neuroinflammatory conditions.

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Figures

Fig 1 Inflammasome components NLRP3, ASC and caspase-1 are expressed in microglia, but not in astrocytes. mRNA and/or protein expression of inflammasome components and substrates NLRP1, NLRP3, NLRC4, ASC (Pycard), Aim-2, Caspase-1, Caspase-4, IL-1α, IL-1β, IL-18 and HMGB1, were studied in mouse microglia, astrocytes, or bone-marrow derived macrophages (BMDM) after 6 hours of stimulation with LPS or Complete Cytokine Mix. RNA was extracted and analyzed for gene expression, relative to L27, by Real-Time PCR (A, C, D). Bar graphs shown represent the mean ± SD. (B) NLRP3, ASC and Caspase-1 expression were analyzed by Western Blot and α-Tubulin was used as loading control. (E) Microglia were analyzed by immunofluorescence for the expression of Iba1, NLRP3, ASC, Caspase-1, HMGB1 and IL-1β, nuclei are stained by DAPI, scale bar: 50μm. Data shown are the mean ± SD of at least three independent experiments. *p<0.05 compared to control (ctrl).
Fig 1 Inflammasome components NLRP3, ASC and caspase-1 are expressed in microglia, but not in astrocytes. mRNA and/or protein expression of inflammasome components and substrates NLRP1, NLRP3, NLRC4, ASC (Pycard), Aim-2, Caspase-1, Caspase-4, IL-1α, IL-1β, IL-18 and HMGB1, were studied in mouse microglia, astrocytes, or bone-marrow derived macrophages (BMDM) after 6 hours of stimulation with LPS or Complete Cytokine Mix. RNA was extracted and analyzed for gene expression, relative to L27, by Real-Time PCR (A, C, D). Bar graphs shown represent the mean ± SD. (B) NLRP3, ASC and Caspase-1 expression were analyzed by Western Blot and α-Tubulin was used as loading control. (E) Microglia were analyzed by immunofluorescence for the expression of Iba1, NLRP3, ASC, Caspase-1, HMGB1 and IL-1β, nuclei are stained by DAPI, scale bar: 50μm. Data shown are the mean ± SD of at least three independent experiments. *p<0.05 compared to control (ctrl).CC BY · PubMed Central
Fig 2 NLRP3 inflammasome activation mechanism in microglia. LPS primed microglia were stimulated with ATP (1 mM, 30 min), Nigericin (Nig, 1.34 μM, 2 h), Monosodium urate (MSU, 100 μg/ml, 5 h) or Aluminium hydroxide (Alum, 300 μg/ml, 5 h). IL-1β production and caspase-1 cleavage were assessed by Western blot (A). Secretion of IL-1β (B), IL-18 (C) and IL-1α(D) in supernatants of wild-type (WT), Nlrp3 -/- and Casp1 -/- microglia was assessed by ELISA. IL-1β secretion was assessed by ELISA in WT and P2rx7 -/- microglia (E) and after treatment with different inhibitors (F-H) (± KCl (25 mM), N-acetyl cystein (NAC, 5 mM), Ca-074Me (10 μM) and cytochalasinD (cytoD, 2 μM)). Inhibitors were added for 30 min prior to inflammasome activation. Data shown are the mean ± SD of at least three independent experiments. *p<0.05, ns = not significant, compared to control (ctrl) (Fig 3B–3E) or compared to no treatment (Fig 3F–3H), #p<0.05, KO compared to WT.
Fig 2 NLRP3 inflammasome activation mechanism in microglia. LPS primed microglia were stimulated with ATP (1 mM, 30 min), Nigericin (Nig, 1.34 μM, 2 h), Monosodium urate (MSU, 100 μg/ml, 5 h) or Aluminium hydroxide (Alum, 300 μg/ml, 5 h). IL-1β production and caspase-1 cleavage were assessed by Western blot (A). Secretion of IL-1β (B), IL-18 (C) and IL-1α(D) in supernatants of wild-type (WT), Nlrp3 -/- and Casp1 -/- microglia was assessed by ELISA. IL-1β secretion was assessed by ELISA in WT and P2rx7 -/- microglia (E) and after treatment with different inhibitors (F-H) (± KCl (25 mM), N-acetyl cystein (NAC, 5 mM), Ca-074Me (10 μM) and cytochalasinD (cytoD, 2 μM)). Inhibitors were added for 30 min prior to inflammasome activation. Data shown are the mean ± SD of at least three independent experiments. *p<0.05, ns = not significant, compared to control (ctrl) (Fig 3B–3E) or compared to no treatment (Fig 3F–3H), #p<0.05, KO compared to WT.CC BY · PubMed Central
Fig 3 Amyloid-β25–35 activates microglia to secrete IL-1β independently from P2X7 signaling. Untreated or LPS primed microglia were stimulated with amyloid-β (Aβ)25–35 (20 or 50 μM) or Aβ35–25 (50 μM) for 5 h. (A) RNA was analyzed for expression of Nlrp3, Il1b and Tnf, relative to L27, by Real-Time PCR (B) Cell free culture supernatants (SN) and cell lysates (XT) were analyzed by Western Blot for expression of caspase-1 and IL-1β. α-Tubulin was used as loading control. (C) IL-1β production in culture supernatants was assessed by ELISA. (D, E) IL-1β production in culture supernatants was assessed by ELISA in wild type, Nlrp3 -/- and Casp1 -/- (D), or P2rx7 -/- (E) microglia. (F) ATP release was quantified in cell supernatant upon treatment. (G) Untreated or LPS primed microglia were stimulated with amyloid-β (Aβ) 1–42 (10μM) for 6h, 24h or 48h. IL-1β production in culture supernatants was assessed by ELISA. (H) Cell free culture supernatants (SN) and cell lysates (XT) were analyzed by Western Blot for expression IL-1β. α-Tubulin was used as loading control (I). Data shown are mean ± SD of at least three independent experiments. *p<0.05 compared to control (ctrl), #p<0.05, KO compared to WT.
Fig 3 Amyloid-β25–35 activates microglia to secrete IL-1β independently from P2X7 signaling. Untreated or LPS primed microglia were stimulated with amyloid-β (Aβ)25–35 (20 or 50 μM) or Aβ35–25 (50 μM) for 5 h. (A) RNA was analyzed for expression of Nlrp3, Il1b and Tnf, relative to L27, by Real-Time PCR (B) Cell free culture supernatants (SN) and cell lysates (XT) were analyzed by Western Blot for expression of caspase-1 and IL-1β. α-Tubulin was used as loading control. (C) IL-1β production in culture supernatants was assessed by ELISA. (D, E) IL-1β production in culture supernatants was assessed by ELISA in wild type, Nlrp3 -/- and Casp1 -/- (D), or P2rx7 -/- (E) microglia. (F) ATP release was quantified in cell supernatant upon treatment. (G) Untreated or LPS primed microglia were stimulated with amyloid-β (Aβ) 1–42 (10μM) for 6h, 24h or 48h. IL-1β production in culture supernatants was assessed by ELISA. (H) Cell free culture supernatants (SN) and cell lysates (XT) were analyzed by Western Blot for expression IL-1β. α-Tubulin was used as loading control (I). Data shown are mean ± SD of at least three independent experiments. *p<0.05 compared to control (ctrl), #p<0.05, KO compared to WT.CC BY · PubMed Central
Fig 4 α-synuclein is not an NLRP3 inflammasome activator. Microglia were stimulated with 5 μM α-synuclein (α-syn), either wild-type (α-syn WT) or mutant (α-syn A53T) (oligomeric form: olig. or fibrillated form: fib.) for 6 h. (A) RNA was extracted and analyzed for expression of Nlrp3, Il1b and Tnf, relative to L27, by Real-Time PCR. (B) After 6 h, 24 h or 48 h of stimulation with α-syn, or 6 h with LPS, cell lysates were analyzed by Western Blot for expression of pro-IL-1β and NLRP3. α-Tubulin was used as loading control. (C) LPS primed microglia were stimulated for 6 h with α-syn or for 30 min with ATP (1 mM) and IL-1β production in culture supernatants was assessed by ELISA. Data shown are the mean ± SD of at least three independent experiments. *p<0.05 compared to control (ctrl).
Fig 4 α-synuclein is not an NLRP3 inflammasome activator. Microglia were stimulated with 5 μM α-synuclein (α-syn), either wild-type (α-syn WT) or mutant (α-syn A53T) (oligomeric form: olig. or fibrillated form: fib.) for 6 h. (A) RNA was extracted and analyzed for expression of Nlrp3, Il1b and Tnf, relative to L27, by Real-Time PCR. (B) After 6 h, 24 h or 48 h of stimulation with α-syn, or 6 h with LPS, cell lysates were analyzed by Western Blot for expression of pro-IL-1β and NLRP3. α-Tubulin was used as loading control. (C) LPS primed microglia were stimulated for 6 h with α-syn or for 30 min with ATP (1 mM) and IL-1β production in culture supernatants was assessed by ELISA. Data shown are the mean ± SD of at least three independent experiments. *p<0.05 compared to control (ctrl).CC BY · PubMed Central
Fig 5 Astrocytes are not able to secrete inflammasome substrates IL-1β and IL-18. Astrocytes were treated for 6h with LPS, P3C, IL-1β, TNFα, IFNγ or CCM and RNA was extracted and analyzed for expression of Il6, Nos2, Cxcl10 (A), Il1b, Nlrp3 and IL18 (B) relative to L27, by Real-Time PCR. CXCL10 secretion was assessed after 6h or 24h of CCM treatment (C). Mouse astrocyte-enriched cultures (AEC-M2) and microglia were primed with Complete Cytokine Mix for (24h) or LPS (6h), respectively, prior to stimulation with ATP (1 mM, 30 min), Nig (1,34 μM, 2h), Aβ25–35 (20 μM, 5h), WT and mutant α-synuclein (5 μM, 5h) or poly(dA:dT) (1 or 2,5 μg/mL, 5h). Levels of IL-1β(D, E and F) and IL-18 (G) in culture supernatants were assessed by ELISA. Data shown are the mean ± SD of at least three independent experiments *p<0.05 compared to control (ctrl).
Fig 5 Astrocytes are not able to secrete inflammasome substrates IL-1β and IL-18. Astrocytes were treated for 6h with LPS, P3C, IL-1β, TNFα, IFNγ or CCM and RNA was extracted and analyzed for expression of Il6, Nos2, Cxcl10 (A), Il1b, Nlrp3 and IL18 (B) relative to L27, by Real-Time PCR. CXCL10 secretion was assessed after 6h or 24h of CCM treatment (C). Mouse astrocyte-enriched cultures (AEC-M2) and microglia were primed with Complete Cytokine Mix for (24h) or LPS (6h), respectively, prior to stimulation with ATP (1 mM, 30 min), Nig (1,34 μM, 2h), Aβ25–35 (20 μM, 5h), WT and mutant α-synuclein (5 μM, 5h) or poly(dA:dT) (1 or 2,5 μg/mL, 5h). Levels of IL-1β(D, E and F) and IL-18 (G) in culture supernatants were assessed by ELISA. Data shown are the mean ± SD of at least three independent experiments *p<0.05 compared to control (ctrl).CC BY · PubMed Central

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Authors
Gustin, A., Kirchmeyer, M., Koncina, E., Felten, P., Losciuto, S., Heurtaux, T., Tardivel, A., Heuschling, P., & Dostert, C.
Affiliations
  • University of Luxembourg (LU)
  • University of Lausanne (CH)
Corresponding author
Dostert, Catherine
ORCID
Venue
PLoS ONE
Publisher
Public Library of Science (PLoS)
Published
2015-06-19
Volume
10
Issue
6
Pages
e0130624
ISSN
1932-6203 (ISSN-L)
DOI
10.1371/journal.pone.0130624
PMC
PMC4474809
OpenAlex
W582182564
Cited by
369
References
55
Citations by year
2026: 21 · 2025: 27 · 2024: 33 · 2023: 38 · 2022: 33 · 2021: 44 · 2020: 59 · 2019: 27 · 2018: 29 · 2017: 37 · 2016: 17 · 2015: 3
FWCI
12.44
Type
journal article
Language
en
Open access
Licence
Locations
  • PLoS ONE (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • IRIS (submittedVersion)
  • Open Repository and Bibliography (University of Luxembourg) (submittedVersion, cc-by-nc-sa)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
  • Figshare (submittedVersion, cc-by)
  • SERVAL (Université de Lausanne) (submittedVersion, other-oa)
Funders
  • Université du Luxembourg
  • Fonds National de la Recherche Luxembourg
  • Université de Lausanne
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Inflammasome and immune disorders
  • Life Sciences › Neuroscience › Neurology › Neuroinflammation and Neurodegeneration Mechanisms
  • Life Sciences › Immunology and Microbiology › Immunology › Immune Response and Inflammation
Keywords
NLRP3 inflammasome, microglia, astrocytes, IL-1β secretion, neuroinflammation, IL-18, IL-1α, alpha-synuclein, amyloid-beta, P2X7 receptor, neurodegenerative diseases
MeSH terms
NLR Family, Pyrin Domain-Containing 3 Protein, Animals, Astrocytes, Brain, Carrier Proteins, Cells, Cultured, Enzyme-Linked Immunosorbent Assay, Mice, Inbred C57BL, Peptide Fragments, Amyloid beta-Peptides, Microglia, Mice, Knockout, Caspase 1, Interleukin-18, Mice, alpha-Synuclein, Interleukin-1alpha, Interleukin-1beta, Receptors, Purinergic P2X7, Inflammasomes

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