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. 2017 Jun 5;8:15556.
doi: 10.1038/ncomms15556.

The PYRIN Domain-Only Protein POP2 Inhibits Inflammasome Priming and Activation

Affiliations
Free PMC article

The PYRIN Domain-Only Protein POP2 Inhibits Inflammasome Priming and Activation

Rojo A Ratsimandresy et al. Nat Commun. .
Free PMC article

Abstract

Inflammasomes are protein platforms linking recognition of microbe, pathogen-associated and damage-associated molecular patterns by cytosolic sensory proteins to caspase-1 activation. Caspase-1 promotes pyroptotic cell death and the maturation and secretion of interleukin (IL)-1β and IL-18, which trigger inflammatory responses to clear infections and initiate wound-healing; however, excessive responses cause inflammatory disease. Inflammasome assembly requires the PYRIN domain (PYD)-containing adaptor ASC, and depends on PYD-PYD interactions. Here we show that the PYD-only protein POP2 inhibits inflammasome assembly by binding to ASC and interfering with the recruitment of ASC to upstream sensors, which prevents caspase-1 activation and cytokine release. POP2 also impairs macrophage priming by inhibiting the activation of non-canonical IκB kinase ɛ and IκBα, and consequently protects from excessive inflammation and acute shock in vivo. Our findings advance our understanding of the complex regulatory mechanisms that maintain a balanced inflammatory response and highlight important differences between individual POP members.

Conflict of interest statement

The authors declare no competing financial interests.

Figures

Figure 1
Figure 1. POP2 expression ameliorates inflammation in vivo.
(a) Immunohistochemical staining of CD68 in red and POP2 in brown in inflamed human lung tissue (left and middle) showing the original magnification × 10 (left, scale bar, 50 μm) and × 40 (middle, scale bar, 10 μm) and human RA synovial lining showing the original magnification × 10 (right, scale bar, 50 μm). (b) Analysis of POP2 expression by flow cytometry in peripheral blood cell populations from WT and POP2TG mice using SmartFlares (n=2). (c) POP2 expression in peripheral blood-derived macrophages isolated from WT and POP2TG mice by immunoblot. Membranes were stripped and re-probed with a β-Actin antibody as loading control (n=5). (d) In vivo imaging of MPO activity of infiltrating neutrophils with a luminescent probe in response to i.p. injection with MSU crystals (3 mg; +) or PBS (−) in WT and POP2TG mice after 4 h (left, scale bar, 1 cm), and quantification of the luminescent counts (right; n=9). (e) Cytokine ELISA and (f) immunoblot for extracellular ASC particles in cleared peritoneal exudates of WT and POP2TG mice 8 h after MSU injection (n=10–13). (g) In vivo imaging of MPO activity correlating to MSU-induced neutrophil infiltration into air pouches 7 h after MSU (3 mg per airpouch; +) or PBS (−) injection in WT and POP2TG mice (left, scale bar, 1 cm) and signal quantification (right; n=9–10). (h) FACS analysis of infiltrating cells in airpouch lavage exudates of WT and POP2TG mice 8 h after MSU injection (n=11). (i) In vivo imaging of MPO activity in LPS-induced peritonitis 4 h after i.p. injection of LPS (2.5 mg kg−1) or PBS (left, scale bar, 1 cm) and quantification (right; n=11). (j,k) Cytokine ELISA and LDH quantification of (j) cleared peritoneal exudates and (k) serum of WT and POP2TG mice 4 h after LPS injection (n=8–19). (l) Survival analysis of the indicated genotypes in response to acute nigericin-induced shock following i.p. injection of nigericin (6 mg kg−1) in LPS-primed mice (0.4 mg kg−1; n=4–17) and significance was determined by asymmetrical log-rank Mantel–Cox survival test. A standard two-tailed unpaired t-test was used for all other calculations. Error bars represent s.e.m., *P<0.05.
Figure 2
Figure 2. POP2 inhibits inflammasome assembly and activation in mouse macrophages.
(a) Interaction of GST-POP2 with endogenous ASC from LPS-primed THP-1 cells and iBMDM TCLs using GST as negative control and showing 10% TCL as input. (b) Immunoblot analysis of ASC polymerization (oligomer) in untreated or LPS/nigericin (Nig)-treated WT BMDM and POP2 BMDM after non-reversible crosslinking of pellets (P) and in TCL. (c,d) Flow cytometric quantification of active caspase-1 in LPS-primed WT, POP2 and Asc−/− BMDM that were treated with control (Ctrl) or (c) nigericin or (d) ATP, % FLICA+ live, single cells is listed. (e) Immunoblot analysis of active caspase-1 p10 and IL-1β release into culture SN of control, LPS-primed and LPS-primed and MSU-treated WT, POP2 and Asc−/− BMDM. Pro-caspase-1 and pro-IL-1β expression in TCL confirms equal loading. ns indicates a cross-reactive nonspecific protein. (fi) Analysis of culture SN for IL-1β release by ELISA in control (Ctrl), LPS-primed (Ctrl+), or LPS-primed and (f) ATP-treated, (g) nigericin-treated or poly(dA:dT)-transfected and (h) MSU, SiO2 or CPPD or (i) TcdB-treated or flagellin (Fla) and Bacillus anthracis lethal toxin-transfected WT, POP2 and Asc−/− BMDM. (h) POP2#1 and POP2#2 BMDM represent two independent POP2TG lines. (jl) Analysis of culture SN for release of (j) IL-18, (k) LDH and (l) IL-1α in response to the treatment of WT, POP2 and Asc−/− BMDM as above. Between three and five independent repeats were performed for each experiment. Significance was calculated by a standard two-tailed unpaired t-test and error bars represent s.e.m., *P<0.05.
Figure 3
Figure 3. POP2 modulates NF-κB-mediated priming of mouse macrophages.
(ac) Analysis of culture SN for (a,c) TNF and (b) IL-6 release by ELISA in response to (a,b) LPS or (c) Pam3CSK4 treatment of WT, POP2 and Asc−/− BMDM for the indicated times. (d,e,g) Immunoblot analysis of phosphorylated (p) proteins and inflammasome proteins in response to LPS treatment of WT and POP2 (d,g) BMDM and (e) iBMDM for the indicated times. Tubulin protein levels were used as a loading control. ns indicates a cross-reactive nonspecific protein. (f) Real-time PCR analysis of Il1b, Il-18, Nlrp3 and Casp1 transcripts in WT and POP2 BMDM in response to LPS treatment for the indicated times presented as fold compared to untreated WT BMDM. Three independent repeats were performed for each experiment. Significance was calculated by a standard two-tailed unpaired t-test and error bars represent s.e.m., *P<0.05.
Figure 4
Figure 4. POP2 inhibits inflammasome activation in human macrophages.
(a,b) Flow cytometric quantification of active caspase-1 in control (Ctrl) and His-POP2 stably expressing THP-1 cells in response to nigericin (Nig) treatment for the indicated times in LPS-primed cells, indicating the % FLICA+ live, single cells showing (a) contour plots and (b) quantification. (c) Kinetic microplate assay of PI uptake by THP-1 cells stably expressing GFP or GFP-POP2 in response to LPS priming followed by nigericin treatment. (d) Real-time PCR analysis of POP2 transcripts in stable control (shRNACtrl) and two independent POP2 shRNA (shRNAPOP2#1 and shRNAPOP2#2)-expressing THP-1 cells presented as fold compared to control shRNA cells. (e) Flow cytometric quantification of active caspase-1 by FLICA assay in LPS-primed POP2-silenced THP-1 cells in response to nigericin (45 min). (fj) Analysis of culture SN for (f,g,i) IL-1β and (h,j) IL-18 release by ELISA in LPS-primed (f) GFP and GFP-POP2-stable THP-1 cells, (g,h) control and His-POP2-stable cells and (i,j) control and POP2 shRNA-expressing THP-1 cells in response to the indicated activators. (k) Primary human macrophages were transfected with a scrambled control or two POP2-specific siRNAs were primed with LPS, and culture SNs were analysed for secreted IL-1β release in response to MSU crystals and ATP. (l) Immunoprecipitation (IP) of ASC or control immunoglobulin G (IgG) from LPS-primed and nigericin-treated WT, control and His-POP2-stable THP-1 cells, followed by immunoblot analysis alongside TCL. HC indicates the antibody heavy chain. Three independent repeats were performed for each experiment. Significance was calculated by a standard two-tailed unpaired t-test and error bars represent s.e.m., *P<0.05.
Figure 5
Figure 5. POP2 inhibits inflammasome priming in human macrophages.
(a,b,d) Analysis of culture SN for IL-6 release by ELISA in response to LPS, MSU crystals and nigericin in (a) GFP and GFP-POP2-stable THP-1 cells, (b) control and His-POP2-stable THP-1 cells and (d) control (shRNACtrl) and POP2 shRNA (shRNAPOP2#1, shRNAPOP2#2) expressing THP-1 cells. (c,ei) Immunoblot analysis of phosphorylated and non-phosphorylated proteins in response to LPS treatment of (c,f) GFP and GFP-POP2 (e,h,i) control and POP2 shRNA-expressing stable THP-1 cells and (g) WT and POP2 BMDM for the indicated times. Tubulin protein levels were used as a loading control and GFP to detect expression of GFP and GFP-POP2. (j) Real-time PCR analysis of the indicated transcripts in control shRNA, POP2#1 shRNA and POP2#2 shRNA-expressing THP-1 cells in response to LPS induction presented as fold compared to untreated control shRNA cells. Three independent repeats were performed for each experiment. Significance was calculated by a standard two-tailed unpaired t-test and error bars represent s.e.m., *P<0.05.
Figure 6
Figure 6. POP2 expression is induced in response to pro- and anti-inflammatory cytokines.
(af) POP2, POP1 and TNF transcripts as indicated were measured by real-time PCR in (a,c,d) primary human macrophages (MΦ). (b,e) THP-1 cells and (f) WT and POP2 BMDM treated with various pro- and anti-inflammatory PAMPs and cytokines for the indicated times. (d,e) Cells were pretreated with LPS or Pam3CSK4 for 1 h and then further incubated in d for a total of 4 h and (e) 4 and 9 h in the presence of the NF-κB inhibitor Bay11-7082. (g) POP2 expression in BMDM isolated from individual POP2TG mice was determined by immunoblot and densitometric quantification and correlated to the release of IL-1β in response to nigericin or the release of TNF in LPS-primed cells and was analysed by linear regression. (h,i) TAT-GFP or TAT-POP2 (0.28 μM) was added to the culture medium of unprimed or LPS-primed (h) BMDM and (i) iBMDM for 30 min followed by 45 min treatment with nigericin. IL-1β release was quantified by ELISA. (j) Proposed function of POP2 as a dual regulator that simultaneously inhibits NF-κB-mediated inflammasome priming and nucleation in macrophages in response to MSU crystals and other activators. POP2 is positioned in a negative feedback loop for inflammasome regulation since it is upregulated by IL-1β, which is blocked upon POP2 expression. For NF-κB signalling POP2 is positioned in a feed-forward regulatory loop since its inhibition of NF-κB leads to POP2 upregulation. Together, POP2 efficiently prevents cytokine release and pyroptosis. Three independent repeats were performed for each experiment in ai. Significance was calculated by a standard two-tailed unpaired t-test, and error bars represent s.e.m., *P<0.05.

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