The innate immune system responds to infection and tissue damage by activating cytosolic sensory complexes called 'inflammasomes'. Cytosolic DNA is sensed by AIM2-like receptors (ALRs) during bacterial and viral infections and in autoimmune diseases. Subsequently, recruitment of the inflammasome adaptor ASC links ALRs to the activation of caspase-1. A controlled immune response is crucial for maintaining homeostasis, but the regulation of ALR inflammasomes is poorly understood. Here we identified the PYRIN domain (PYD)-only protein POP3, which competes with ASC for recruitment to ALRs, as an inhibitor of DNA virus–induced activation of ALR inflammasomes in vivo. Data obtained with a mouse model with macrophage-specific POP3 expression emphasize the importance of the regulation of ALR inflammasomes in monocytes and macrophages.
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We thank B.C. Schaefer (Uniformed Services University of the Health Sciences) for the promoter of the gene encoding ubiquitin C; J. DeGregori (University of Colorado Health Sciences Center) for the plasmid pLXS-hCARΔcyt; D. Trono (École Polytechnique Fédérale de Lausanne) for plasmids pMD2.G and psPAX2; K.A. Fitzgerald (University of Massachusetts) for Aim2−/− mice; the Northwestern University Transgenic and Targeted Mutagenesis Laboratory for assistance in generating transgenic mice; and A.D. Radian for the isolation of human macrophages. Supported by the US National Institutes of Health (GM071723, HL097183, AI092490, AI082406, AI099009 and AR064349 to C.S.; AR057532 to A.D., AR050250, AR054796, AI092490 and HL108795 to H.P.; AR064313 to C.M.C.; and T32AR007611 to L.d.A.), the National Cancer Institute (CA060553), the National Institute of Arthritis and Musculoskeletal and Skin Diseases (AR057216), the American Heart Association (12GRNT12080035 to C.S.), the Arthritis Foundation (AF161715 to S.K.), the American Heart Association (11POST585000 to L.d.A.) and the Solovy/Arthritis Research Society (H.P.).
The authors declare no competing financial interests.
Integrated supplementary information
(a) cDNA showing the open reading frame of POP3 (Genbank accession number: KF562078) (b) A nucleotide BLAST (blastn) analysis against the assembled human RefSeq genomes (http://blast.ncbi.nlm.nih.gov) detailing the genomic location of POP3 within the HIN-200 cluster flanked by IFI16 and PYHIN1 on human chromosome 1q23.
(a) Amino acid sequence of POP3. The PYD is shaded grey. The predicted α-helices are marked with blue lines (bottom), while the corresponding α-helices of AIM2, as determined by crystal structure1, are marked with a red line (top). (b) ClustalW alignment of the amino acid sequences corresponding to the PYDs of POP3 and human HIN-200 members. (c) ClustalW alignment of all human PYDs. The characteristic amino acid motifs found in HIN-200 members, which are also present in POP3, are highlighted in yellow. (d) Phylogenetic tree cluster analysis of sequences used in b. The HIN-200 cluster, which includes POP3 is highlighted in yellow.
(a, c, d) hMΦ were transfected with either control or a, POP3#2 or c, d, POP3 siRNAs and infected with MVA or transfected with poly(dA:dT) as indicated for 16 h and analyzed for a, mature IL-1β and c, IL-6 by ELISA (n = 3 ± s.e.m.) and d, TCL from Figure 3f, were analyzed in parallel for expression of AIM2 and IFI16 by immunoblot. (b, f) THP-1 cells were transfected with siRNAs as above, and infected with MVA, transfected with poly(dA:dT) or treated with MSU or SiO2, as indicated for 16 h and analyzed for b, IL-1β secretion and f, IFN-b by ELISA (n = 3 ± s.e.m.). (e, g) THP-1 (GFP) and THP-1 (GFP-POP3) cells were analyzed for secretion of e, TNFα and g, IFN-β in response to MVA and MCMV infection, transfection of poly(dA:dT) and treatment with MSU as indicated by ELISA (n = 3 ± s.e.m.). (h) The POP3 antibody does not cross-react with other POP family members. HEK293 cells were transfected with Myc-tagged POP1, POP2 and POP3 and immunoprobed with our custom POP1, POP2 and POP3-specific antibodies. (i) The POP3 antibody does not cross-react with the related PYDs of AIM2 and IFI16. HEK293 cells were transfected with GFP or RFP-tagged POP3, AIM2-PYD and IFI16-PYD and immunoprobed with our POP3 antibody and with GFP and RFP antibodies as control. * denotes a cross-reactive protein. Data are representative of 3 experiments (a), 2 experiments (b-d, f) and 1 experiment (e, g-i). (a) *P<0.0001, **P=0.0049; (b) *P=0.0199, **P=0.0495; (f) *P<0.0001; (g) *P=0.0031, **P=0.0161, ***P=0.0013;.
Supplementary Figure 4 Gating strategy for immunophenotyping of peripheral blood and peritoneal lavage cells.
(a) Peripheral blood cells and (b) peritoneal lavage cells obtained 6 h after MCMV infection were gated according to established cell surface markers, as indicated.
(a) Thioglycollate-elicited PM were isolated by peritoneal lavage, transfected with poly(dA:dT) for 16 h and analyzed for mature IL-1β by ELISA (n = 3 ± s.e.m.). (b) BMDM isolated from a 2nd line of CD68-POP3 TG mice were infected with MVA, treated with LPS/ATP or transfected with poly(dA:dT) for 16 h and analyzed for mature IL-1β by ELISA (n = 3 ± s.e.m.). (c) BMDM of UbiC-hCAR TG mice were immunoprobed for expression of hCARΔcyt using HEK293 cells transiently transfected with hCARΔcyt as a control. (d) WT (top panel) and UbiC-hCAR TG (bottom panel) BMDM were infected with increasing MOI of a GFP-expressing AdV and analyzed by fluorescence and phase contrast microscopy. (e) UbiC-hCAR TG BMDM were infected with low MOI of AdV expressing GFP or GFP-POP3 and transfected 48 h later with poly(dA:dT) or infected with MVA for 16 h and analyzed for secreted IL-1β by ELISA. (f) WT and POP3 transgenic BMDM were infected with MVA and analysed for mRNA expression of IL1ra and Il18bp (n=3 ± s.e.m.). Data are representative of two (a-c, e) and one (d, f) experiments. (b) ***P=0.0097, **P=0.0038; (e) ***P=0.0001, **P=0.0058; (f) *P=0.0029 (Il1ra); *P=0.0009 (Il18bp).
Splenocytes obtained 36 h after MCMV infection were gated according to established cell surface markers, as indicated.
(a) WT and CD68-POP3 TG mice were i.p. injected with PBS or MSU crystals (10 mg/mouse) and mice were imaged for MPO activity in vivo 5 h later (n=3-7), showing representative examples. (b) Model of the type I IFN-induced regulatory loop of cytosolic DNA-induced inflammasome response that involves POP3. (a) Data are representative of one experiment.
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Khare, S., Ratsimandresy, R., de Almeida, L. et al. The PYRIN domain–only protein POP3 inhibits ALR inflammasomes and regulates responses to infection with DNA viruses. Nat Immunol 15, 343–353 (2014). https://doi.org/10.1038/ni.2829
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