NLRP12/Monarch-1 Suppression of the NLR Gene Blimp-1/PRDM1

Blimp-1/PRDM1 Mediates Transcriptional Suppression of the NLR Gene NLRP12/Monarch-1

This information is current as Christopher A. Lord, David Savitsky, Raquel Sitcheran, of September 29, 2021. Kathryn Calame, Jo Rae Wright, Jenny Pan-Yun Ting and Kristi L. Williams J Immunol 2009; 182:2948-2958; ; doi: 10.4049/jimmunol.0801692

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2009 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Blimp-1/PRDM1 Mediates Transcriptional Suppression of the NLR Gene NLRP12/Monarch-11

Christopher A. Lord,* David Savitsky,2§ Raquel Sitcheran,‡ Kathryn Calame,§ Jo Rae Wright,* Jenny Pan-Yun Ting,‡ and Kristi L. Williams3*†

NLR (nucleotide-binding domain, leucine-rich repeat) proteins are intracellular regulators of host defense and immunity. One NLR gene, NLRP12 (NLR family, pyrin domain containing 12)/Monarch-1, has emerged as an important inhibitor of inflammatory gene expression in human myeloid cells. This is supported by genetic analysis linking the loss of a functional NLRP12 protein to hereditary periodic fever. NLRP12 transcription is diminished by specific TLR stimulation and myeloid cell maturation, consistent with its role as a negative regulator of inflammation. The NLRP12 promoter contains a novel Blimp-1 (B lymphocyte-induced maturation protein-1)/PRDM1 (PR domain-containing 1, with ZNF domain) binding site, and Blimp-1 reduces NLRP12 promoter activity, expression, and histone 3 acetylation. Blimp-1 associates with the endogenous NLRP12 promoter in a TLR-inducible Downloaded from manner and mediates the down-regulation of NLRP12 expression by TLR agonists. As expected, the expression of NLRP12 and Blimp-1 is inversely correlated. Analysis of Blimp-1؊/؊ murine myeloid cells provides physiologic evidence that Blimp-1 reduces NLRP12 gene expression during cell differentiation. This demonstrates a novel role for Blimp-1 in the regulation of an NLR gene. The Journal of Immunology, 2009, 182: 2948–2958.

he recognition of microbial components during host in- also known as CATERPILLER, NOD-LRR, NACHT-LRR,or http://www.jimmunol.org/ fection is a key step in activating the innate immune re- NOD-like receptor (5–8). Mutations in several NLR genes have T sponse followed by the induction of inflammatory gene been associated with human disease states, including autoimmu- expression. Pattern-recognition receptors exemplified by TLRs are nity and inflammation. Members of the NLR gene family are in- key regulators in host response to bacterial, viral, and fungal com- volved in regulating cellular activation after exposure to specific or ponents (1, 2). Central to this response is the ability to up-regulate multiple pathogen-derived products (9). NOD2 mediates cellular genes involved in host protection, innate and adaptive immune cell responses to peptidoglycan-derived muramyl dipeptide, leading to recruitment, and pathogen clearance. Although the TLR-mediated activation of inflammatory cytokines and the release of antimicro- inflammatory response is critical for providing immune defense bial peptides from cytosolic granules (10–12). Nlrp1b/Nalp1b reg- by guest on September 29, 2021 against pathogens, dysfunctional responses may lead to both acute ulates disease susceptibility of some murine strains to anthrax le- and chronic inflammatory states manifested as sepsis, inflamma- thal toxin (13). NLRC4/IPAF mediates caspase-1 and IL-1 tion, and autoimmunity (3). Therefore, the intensity and duration processing in response to the flagellin of Salmonella typhimurium of TLR responses must be tightly controlled. and Legionella pneumophila (14–16). Naip5 mediates host sus- In addition to TLR, recent research has focused on the discovery ceptibility to the intracellular pathogen Legionella pneumophila and characterization of a new immune gene family, the NLR (nu- (17). NLRP3/cryopyrin/NALP3 mediates caspase-1 and IL-1 pro- 4 cleotide-binding domain, leucine-rich repeat) gene family (4), cessing in response to an array of stimuli (18–21). The more recently described NLRX1 gene negatively regulates the intracellular type I IFN signaling pathway in mitochondria (22). Taken to- Departments of *Cell Biology and †Immunology, Duke University Medical Center, gether, these data suggest that the NLR genes are involved in reg- Durham NC 27710; ‡Lineberger Comprehensive Cancer Center, the University of North Carolina at Chapel Hill, Chapel Hill, NC 27599; and §Departments of Micro- ulating a variety of host defense processes. biology and Biochemistry & Molecular Biophysics, Columbia University College of The relevance of NLR genes is most apparently revealed by the Physicians and Surgeons, New York, NY 10032 genetic analysis of patients suffering from immune and inflamma- Received for publication May 27, 2008. Accepted for publication December 19, 2008. tory disorders. Mutations in CIITA result in the immunodeficiency The costs of publication of this article were defrayed in part by the payment of page type II bare lymphocyte syndrome (group A) (23). NOD2 muta- charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. tions are associated with increased susceptibility to inflammatory 1 This work was supported by National Institutes of Health Grants HL-30923 and Crohn’s disease and to a granulomatous disorder known as Blau HL-084917 (to J.R.W.) and AI057175 and AI063031 (to J.P.T.), as well as a National syndrome (24–26). Hyperactive mutants of the NLRP3 (CIAS1) Research Service Award, University of North Carolina Center for AIDS Research, gene predispose patients to a variety of autoinflammatory disorders Amgen/Federation of Clinical Immunology Societies Fellowship Award, Southeast Regional Center of Excellence for Emerging Infections and Biodefense, and support classified as cryopyrin-associated periodic syndrome, which falls from the American Cancer Society (to K.L.W.). under the general classification of hereditary periodic fever (HPF) 2 Current address: Department of Dermatology, Asahikawa Medical College, Asa- (27–32). Mutations in NLRP1/NALP1 have been genetically linked hikawa, Japan. 3 Address correspondence and reprint requests to Dr. Kristi L. Williams, Department of Cell Biology, Box 3709, Duke University Medical Center, Durham, NC 27710. E-mail address: [email protected] ZNF domain; HKLM, heat-killed Listeria monocytogenes; qPCR, quantitative PCR; 4 Abbreviations used in this paper: NLR, nucleotide-binding domain, leucine-rich DAPA, DNA affinity purification assay; ChIP, chromatin immunoprecipitation; DRB, repeat; HPF, hereditary periodic fever; NLRP12, NLR family, pyrin domain contain- 5,6-dichlororibofuranosyl benzimidazole; GusB, ␤-glucuronidase. ing 12; IRAK, IL-1R-associated kinase; NIK, NF-␬B-inducing kinase; Blimp-1, B lymphocyte-induced maturation protein-1; PRDM1, PR domain-containing 1, with Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00 www.jimmunol.org/cgi/doi/10.4049/jimmunol.0801692 The Journal of Immunology 2949 with autoimmune disease (33), while NLRP7/NALP7 has been as- data provide a new linkage between Blimp-1 and the innate im- sociated with hydatidiform mole (34). These studies underscore mune TLR and NLR pathways. the contention that NLR family members are important players in both the maintenance of normal immune responses and the onset Materials and Methods of inflammatory disorders. Mice We and others have identified an NLR family member Mon- Ϫ ϩ Prdm1flox/flox and prdm1flox/ mice (48) were crossed with Rosa26ERCre/ arch-1/PYPAF7, recently renamed NLRP12 (NLR family, pyrin mice to generate controls (Rosa26ϩ/ϩprdm1flox/flox) and experimental domain containing 12) (35–37). NLRP12 is expressed primarily by (Rosa26ERCre/ϩprdm1flox/flox) mice. Mice were injected with 1.5 ml of 10 cells of the myeloid-monocytic lineage, including monocytes, mg/ml tamoxifen in sunflower seed oil (0.5 ml/day for 3 days) to induce granulocytes, and eosinophils in humans (36). Although it has gene deletion 3 wk before tissue harvest. Mice were housed in the barrier facility of Columbia University (New York, NY). All experiments were been suggested that some NLR proteins are involved in “sensing” approved by the Institutional Animal Care and Use Committee of Colum- microbial components, currently there is no known ligand for NLR bia University. proteins in general and for NLRP12 specifically. In vitro studies in human cells employing both gene transfection and small hetero- Reagents duplex RNA-mediated gene silencing suggest that NLRP12 func- LPS derived from Escherichia coli 011:B5 (Sigma-Aldrich) was used at a tions as a negative regulator of TLR- and TNFR-induced NF-␬B final concentration of 100 ng/ml; PMA (Sigma-Aldrich) was used to dif- signaling in human cells. NLRP12 blocks IRAK (IL-1R-associated ferentiate cells at a concentration of 50 ng/ml. Abs included anti-Flag M2 (Sigma-Aldrich), anti-p65 (Rockland), anti-Blimp-1 (Abcam), anti- kinase)-1 hyperphosphorylation/activation (37) and facilitates the acetylated-H3 (Upstate Biotechnology), and mouse IgG (Upstate degradation of NF-␬B-inducing kinase (NIK), leading to reduced Biotechnology). Downloaded from NF-␬B activation (38). A recent study of patients with the clinical manifestation of HPF but without mutations in several HPF-linked Tissue culture cells and conditions genes revealed a new genetic link to NLRP12 (39). In contrast to HEK293T cells, undifferentiated HL-60 cells, undifferentiated U937 cells, the genetic linkage of NLRP3 and cryopyrin-associated periodic and primary adherent blood cells were maintained as previously described (37). All cells were grown at 37°C with 5% CO . HL-60 cells were dif- syndrome wherein a variety of hyperactive, single nucleotide mis- 2 ferentiated with 1.25% DMSO as described below. U927 cells were stim- sense mutants have been found in patients, the known disease- ulated with 100 ng/ml LPS or differentiated with 50 ng/ml PMA. http://www.jimmunol.org/ linked NLRP12 mutants are represented by a splicing variant that caused an 11-residue insertion and truncation of the LRR region in Primary cell isolation and stimulation one family, and a nonsense mutation that truncated a portion of the Human granulocytes were isolated from buffy coats (American Red Cross) NBD domain and all of the LRR region in a second family (39). using the Lymphocyte Separation Media (ICN Pharmaceuticals). For ad- These truncated NLRP12 proteins failed to inhibit NF-␬B activa- herent cell purification, cells were plated and allowed to adhere for2hat tion, supporting the conclusion that NLRP12 exhibits an inhibitory 37°C. After three washes with PBS to remove nonadherent cells, adherent cells were stimulated for the indicated time points with 100 ng/ml LPS function. from E. coli (LPS 011:B5; Sigma-Aldrich) or, where indicated, 100 ng/ml In contrast to most innate immune genes, human NLRP12 ex- K12 LPS, 0.1 ␮g/ml Pam3Cys, 1 ϫ 108 cells/ml heat-killed Listeria mono- pression is down-regulated after activation of myeloid cells with cytogenes (HKLM), or 1 ␮g/ml flagellin (InvivoGen). by guest on September 29, 2021 M. tuberculosis either TLR2 or TLR4 agonists, , or exposure of Isolation and differentiation of murine bone marrow cells cells to TNF-␣ or IFN-␥ (36, 37). Based on our present under- NLRP12 Bone marrow cells from either wild-type C57BL/6N or treated standing of the function of , we hypothesize that NLRP12 ERCre/ϩ flox/flox ϩ/ϩ flox/flox is present before immune stimulation to maintain a quiescent phe- Rosa26 prdm1 and Rosa26 prdm1 mice were flushed from the tibiae and femurs and cultured at 1 ϫ 106 cells/ml in MEM notype, but during infection its expression is reduced to allow for medium supplemented with 5% GM-CSF (culture supernatant from X63 a full inflammatory response to occur. At the center of this hy- cells transfected with murine GM-CSF cDNA (provided by Dr. Chris Nic- pothesis is the down-regulation of NLRP12 by TLR and during chitta, Duke University, Durham, NC)). Total cells (adherent and nonad- states of myeloid cell differentiation. However, the mechanism by herent) were isolated at the indicated time points, and RNA was prepared using TRIzol reagent (Invitrogen) and analyzed for NLRP12 expression by which TLRs and myeloid cell differentiation processes regulate quantitative PCR (qPCR). NLR gene expression is an uncharted territory. In this work, we show that the transcriptional silencer Blimp-1 (B lymphocyte-in- Expression plasmids duced maturation protein-1)/PRDM1 (PR domain-containing 1, The Flag-tagged Blimp-1 expression vector was previously described (49). with ZNF domain) is involved in the transcriptional repression of A sequence containing 1158 bp of the NLRP12 promoter was cloned into NLRP12 after TLR activation or after myeloid cell differentiation. pGL3-Basic (Promega) luciferase reporter vector and called NLRP12 luc. Blimp-1 is a DNA-binding factor that recruits histone deacety- Site-directed mutagenesis was used to mutate four base pairs of the Blimp-1 binding site in using the QuikChange site-directed mutagenesis kit lases, histone lysine methyltransferases, histone arginine methyl- (Stratagene). Primers were NLRP12 5Ј-GGAAGAGACAGCAGACTC transerses, and corepressors to induce promoter silencing (40–43). GAGAATCTTTTTCATC-3Ј and 5Ј-GATGAAAAAGATTCTCCTCTCT The role for Blimp-1 in regulating B cell differentiation into Ab- GCTGTCTCTTCC-3Ј. To generate the pGL4.10-based luciferase vectors producing plasma cells has received the most attention (44). How- the following forward primer sequences were used, each containing KpnI Ј ever, the role of Blimp-1 extends significantly beyond B cell dif- sites for cloning: pGL4-1412, 5 -GCGGTACCGAGAATTAGCCAGG CGTGTTG-3Ј; pGL4–852, 5Ј-GCGGTACCGCATGGTGGCGCA ferentiation, as it also controls T cell homeostasis and activation TGTCTGTGGT-3Ј;pGL4–399,5Ј-GCGGTACCCTGGAAGAGGCGCAG (45, 46). Most relevant to this work, Blimp-1 expression is induced TCGCAGTT-3Ј; a common reverse primer was used for all PCR reactions during differentiation of myeloid cell lines, whereby it reduces and contains a HindIII site for cloning: 5Ј-GCGAAGCTTC Ј c-myc expression and promotes the up-regulation of myeloid- CTGGAGGCTGAGATGCTCCTATG-3 . monocytic markers CD11b and CD11c (47). Identification of a potential Blimp-1 binding site in the NLRP12 In this report we identify Blimp-1 as a key regulator of TLR- promoter mediated and differentiation-associated reduction of NLRP12 in The National Center for Biotechnology Information and Transcription El- human myeloid-monocytic cells. We propose that Blimp-1 is im- ement Search Software (TESS) (www.cbil.upenn.edu/tess/) databases were portant in restricting NLRP12 expression, thereby modulating the used to search for potential transcription factor binding sites within the extent by which NLRP12 inhibits inflammatory signaling. These NLRP12 promoter. 2950 Blimp-1 DOWN-REGULATES NLRP12

Luciferase reporter gene assays trols and are expressed in relative numbers. Primers were: human NLRP12 forward, 5Ј-AGAGGACCTGGTGAGGGATAC-3Ј, reverse, 5Ј-CTTCCA ϫ 6 For transfections, 1 10 U937 cells were transfected with Amaxa GAAGGCATGTTGAC-3Ј, probe, 5Ј- CCCGTCCTCACTTGGGAACCA- ␮ ␮ ␬ Nucleofector (Amaxa) with 0.2 g of phRL-luc, 0.5 gofNF- Bluc, 3Ј; 18S forward, 5Ј-GCTGCTGGCACCAGACTT-3Ј, reverse, 5Ј-CGGC NLRP12luc, or mutNLRP12luc. Where indicated, cells were transfected TACCACATCCAAGG-3Ј, probe, 5Ј-CAAATTACCCACTCCCGACC ␮ with 1.0 g of Flag-Blimp-1 or stimulated with LPS. Firefly luciferase CG-3Ј; GusB (Applied Biosystems: Hs99999908_m1); mouse NLRP12 values were normalized to Renilla luciferase control values when harvested forward, 5Ј-CGACCCACCAGAACCTTCAG-3Ј, reverse, 5Ј-CAGG 24 h posttransfection. CAGCATGTTCCTTTCC-3Ј, probe, 5Ј-TCCAGACTCAGTCCACA TACTTAC-3Ј. EMSA HEK293T cells were transfected with pcDNA3 or a Flag-tagged Blimp-1 Results using FuGENE 6 (Roche). EMSA was performed as previously described NLRP12 expression is regulated by specific TLRs (50). For supershifts, 1 ␮l of anti-Flag M2 Ab (Sigma-Aldrich) or anti-p65 Ab (Rockland) was added and the binding reaction was allowed to proceed NLRP12 is expressed by cells of the myeloid and monocytic lin- for an additional 15 min. Protein-DNA complexes were resolved on a eage (35, 37). Myeloid and monocytic cells are involved in the nondenaturing polyacrylamide gel. Oligo pairs were annealed in STE innate immune response to pathogens, and recognize invading mi- buffer (10 mM Tris (pH 8.0), 50 mM NaCl, 1 mM EDTA) and T4 kinase- labeled with [␣-32P]cytidine 5Ј-triphosphate. Primers used for NLRP12 croorganisms via TLRs on their cell surface (53). We have recently EMSA were 5Ј-GGAAGAGACAGCAGAAGTGAAAATCTTTTTCA shown that NLRP12 is a negative regulator of TLR1/2 and TLR4 TC-3Ј and 5Ј-GATGAAAAAGATTTTCACTTCTGCTGTCTCTTCC-3Ј. signaling in human monocytic-macrophage cell lines, and that ab- The consensus Blimp-1 binding site is underlined. lation of NLRP12 increases proinflammatory gene expression (37).

DNA affinity purification assays (DAPA) Moreover, NLRP12 inhibits CD40-mediated NF-␬B activation by Downloaded from promoting the protein degradation of NIK (38). NLRP12 mRNA ␮ DAPA experiments were performed using the MACS streptavidin kit expression is reduced within1hofTLR1/2, TLR4, or microbial (Miltenyi Biotec). Briefly, 1 ␮gof5Ј-biotinylated, duplexed oligo corresponding to the Blimp-1 binding site in the NLRP12 promoter (shown pathogen stimulation (37) and to a lesser extent after exposure of above for EMSA) was coupled to streptavidin beads for 30 min at 4°C cells to TNF-␣ or IFN-␥ (36). To determine whether NLRP12 followed by addition of nuclear extract (500 ng) for 30 min. DNA-protein expression is regulated by other TLR agonists, we exposed pri- complexes were immobilized magnetically and washed four times with 20

mary human granulocytes for1htoHKLM (TLR2), the synthetic http://www.jimmunol.org/ mM HEPES-KOH (pH 7.9), 100 mM KCl, and 2.5 mM MgCl2. Specifi- cally bound proteins were eluted with 20 mM HEPES-KOH (pH 7.9) and analog of dsRNA (poly(I:C)) (TLR3), E. coli K12 rough LPS 1 M NaCl. Eluates were electrophoresed on denaturing polyacrylamide (TLR4), and S. thyphimurium flagellin (TLR5) (Fig. 1A). gels for subsequent immunoblot analysis using anti-Blimp-1. Pam3Cys4 (TLR1/2) was used as a control, as we have previously observed that NLRP12 expression is down-regulated in response to Chromatin immunoprecipitation (ChIP) assays stimulation with this agonist. NLRP12 mRNA expression was dra- ChIP analysis was performed as previously described (51) with some mod- matically reduced after exposure of granulocytes to Pam3Cys4, ifications. Briefly, protein-DNA complexes were cross-linked for 10 min HKLM, and K12 LPS, but not in response to stimulation with with formaldehyde and cells were harvested. Cells were either snap-frozen in dry ice/EtOH and stored at Ϫ80°C or immediately used. DNA in lysed poly(I:C) or flagellin, although these cells were determined to ex- cells was sonicated to lengths of 500-1000 bp, and anti-Blimp-1 (Abcam), press and be responsive to TLR3 and TLR5 stimulation (Fig. 1, A by guest on September 29, 2021 anti-acetylated-H3 (Upstate Biotechnology) or mouse IgG (Upstate Bio- and B). Functional TLR3 and TLR5 receptors have been previ- technology) was used for immunoprecipitation overnight at 4°C. SYBR ously observed in granulocytes (54, 55). The observation that Green qPCR was performed and values were expressed as a percentage of NLRP12 expression was not altered to the same extent in response total input DNA for each time point. Primers to amplify the NLRP12 promoter were: 5Ј-CCTGGAAGAGGCGCAGTCGCAGTT-3Ј (forward) and to each TLR agoinst suggests that down-regulation of NLRP12 is 5Ј-CCGAGGAGCCACTTGGCTGATGAA-3Ј (reverse). For visualiza- mediated by specific TLRs. tion, PCR fragments from the ChIP analysis were run on an agarose gel. To assess the kinetics of NLRP12 down-regulation by TLR Immunoprecipitation and Western analysis stimulation, human primary adherent peripheral blood mononuclear cells were isolated and exposed to purified LPS from E. coli, The U937 cell line was stimulated with 100 ng/ml LPS or 50 ng/ml PMA which activates TLR4 (Fig. 1C). NLRP12 expression was down- for the indicated time. Cells were lysed in lysis buffer (50 mM Tris-HCl regulated 1 h after TLR stimulation, and it remained low 6, 8, and (pH 8.0), 150 mM NaCl, 1% Nonidet P-40) supplemented with complete protease inhibitor cocktail (Roche). Protein concentrations were deter- 16 h poststimulation. This indicates the sustained down-regulation mined by Bradford assay (Bio-Rad), and equivalent amounts of cellular of NLRP12 in primary human myeloid cells via TLR. extract were used in subsequent immunoprecipitations using rabbit polyclonal antisera against human NLRP12 as described with polyclonal goat TLR stimulation affects NLRP12 mRNA transcription anti-Blimp-1 (Abcam) or mouse monoclonal anti-GAPDH (Chemicon In- ternational). Immunoblots were visualized by ECL (Pierce). The sustained decrease in NLRP12 expression following TLR stimulation led us to examine whether NLRP12 is regulated at the Measurement of NLRP12 mRNA stability level of mRNA stability, transcription, or both. Nuclear vs cytoplasmic transcripts were isolated using the PARIS kit (Am- We first examined the transcription rate of the NLRP12 gene by bion) following the manufacturer’s protocol, and qPCR was performed comparing nuclear and cytoplasmic NLRP12 mRNA levels after with primers recognizing process mRNA. Measurement of DRB (5,6-di- exposure of U937 cells to LPS (Fig. 1D). This approach has been chlororibofuranosyl benzimidazole)-regulated mRNA stability was performed as previously described (52). HL-60 cells were treated with 1.25% proven as a sensitive and reliable method often used as an alter- DMSO for 4 days to induce differentiation to neutrophils and expression of native to a nuclear run-on assay (52). U937 cells were stimulated NLRP12. Cells were replated and stimulated with LPS, exposed to 50 ␮M for the indicated time points with LPS followed by separate iso- DRB (Sigma-Aldrich), dissolved in 0.1% DMSO, or exposed to 0.1% lation of nuclear and cytoplasmic mRNA followed by qPCR anal- DMSO as a vehicle control. NLRP12 expression was quantified by qPCR ysis of NLRP12 transcripts. The levels of NLRP12 mRNA were and normalized to the housekeeping gene ␤-glucuronidase (GusB) expression levels. similar in both the nuclear and cytoplasmic compartments, suggesting that the reduction of NLRP12 mRNA caused by TLR sig- RNA preparation and qPCR naling is partly or primarily attributed to an effect on transcription. Total RNA was isolated according using TRIzol reagent (Invitrogen). As an alternative measure of mRNA stability, we used DRB to qPCR results are normalized to 18S rRNA or GusB mRNA internal con- chemically block gene transcription in the HL-60 cell line. The The Journal of Immunology 2951

A C 1.80 1. 2 0 1.60 1. 0 0 1.40 1.20 0.80 1.00 0.60 0.80 0.60 0.40

0.40 Relative NLRP12 mRNA mRNA NLRP12 Relative

Relative NLRP12 mRNA mRNA NLRP12 Relative 0.20 0.20

Control Pam3Cys HKLM Poly I:C K12 LPS Flagellin 1 hr 6 hrs 8 hrs 16 hrs TLR1/2 TLR2 TLR3 TLR4 TLR5

B D 1.20 4500 cytoplasmic 1.00 nuclear 3500 0.80

2500 Downloaded from 0.60

1500 0.40 Relative NLRP12 mRNA mRNA NLRP12 Relative

Relative TLR3 mRNA TLR3 Relative mRNA 0.20 500

granulocytes HEK 293T Total PBMCs 0 hr 3 hr 8 hrs 24 hrs http://www.jimmunol.org/

DMSO 25000 E LPS DRB 1.40 DRB+LPS 20000 1.20

1.00 15000 0.80 by guest on September 29, 2021 10000 0.60

0.40 Relative TLR5 mRNA TLR5 Relative mRNA

5000 mRNA NLRP12 Relative 0.20

granulocytes HEK 293T Total PBMCs 0 hr 1 hr 2 hrs 3 hrs 4 hrs FIGURE 1. NLRP12 expression is regulated by specific TLRs. A, Human primary granulocytes were stimulated with the indicated TLR agonists for 1 h. NLRP12 expression was analyzed by qPCR analysis of total RNA. NLRP12 expression was normalized to the housekeeping gene GusB. Data represent an average of three separate experiments. B, TLR3 and TLR5 expression in total human peripheral blood cells, granulocytes isolated from human peripheral blood cells by Ficoll separation, and in the HEK293T cell line were assessed by qPCR. TLR expression was normalized to the expression of GusB. PMBCs indicate peripheral blood mononuclear cells. C, NLRP12 expression in human adherent peripheral blood cells stimulated for 1, 6, 8, and 16 h with phenol-purified LPS (TLR4 agonist). NLRP12 expression was normalized to the expression of 18S rRNA and represented as fold difference compared with control. Error bars represent the SEM of three separate cell preparations and experiments. D, The reduction of nuclear verses cytoplasmic NLRP12 expression after TLR activation. U937 cells were stimulated with 500 ng/ml LPS for 3, 5, 8, 18, and 24 h followed by isolation of nuclear vs cytoplasmic mRNA as described in Materials and Methods. NLRP12 mRNA abundance was then quantified by qPCR and normalized to GusB. E, The stability of the NLRP12 mRNA is similar in cells treated with a transcriptional inhibitor or stimulated with LPS. The human pre-monocyte cell line HL-60 was treated with 1.25% DMSO for 3 days to induce differentiation into NLRP12- expressing neutrophils. Differentiated HL-60 cells were then stimulated with LPS (f), treated with DRB (ࡗ), treated with DRB plus stimulated with LPS (Œ, or exposed to 0.1% DMSO (F) as a vehicle control for 0, 1, 2, 3, and 4 h. NLRP12 mRNA abundance was then quantified by qPCR. The amount of NLRP12 mRNA at the time before DRB addition (0 h) was set to 1, and the amount remaining at the indicated time points was determined. Error bars represent the SEM of five separate experiments.

human monocyte HL-60 cell line was treated with DMSO for 3 the untreated control cells; however, its level was reduced in cells days to promote granulocyte differentiation and NLRP12 expres- treated with the transcriptional inhibitor DRB or with LPS alone. sion. It was necessary to use the HL-60 cell line, as addition of The reduction with these two treatments is not statistically different DMSO to the U937 cell line promotes differentiation-induced as assessed by a Student’s t test ( p ϭ 0.17). The inclusion of both down-regulation of NLRP12 (K. L. Williams, unpublished obser- DRB and LPS did not cause an additive effect. Taken together, vations). Differentiated HL-60 neutrophils were then analyzed for these results suggest that the decline in NLRP12 mRNA is signif- the level of NLRP12 transcripts after TLR stimulation, blocking icantly altered by the transcriptional inhibitor DRB, and that the transcription with DRB in DMSO, with both LPS and DRB or with reduction of NLRP12 mRNA caused by TLR signaling is attrib- DMSO alone (Fig. 1E). NLRP12 mRNA was relatively stable in uted to an effect on transcription. 2952 Blimp-1 DOWN-REGULATES NLRP12 Downloaded from http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 2. The NLRP12 promoter contains a potential Blimp-1 binding site. A, Sequence of the human NLRP12 promoter region from Ϫ399 to ϩ223 bp. Several transcription factor DNA binding sites are shown. The DNA binding site for Blimp-1 is underlined. The transcriptional start site is denoted by an arrow (ϩ1), and the ATG of the translational start site is underlined. B, Comparison of the putative Blimp-1 binding site in the NLRP12 promoter with the Blimp-1 DNA binding sequences of the 10 other Blimp-1 targets (boxed). C, U937 human monocyte cells were stimulated with LPS for 0, 2, 4, 8, and 24 h. NLRP12 protein was detected by immunoprecipitation followed by immunoblot with two different NLRP12 Abs as described. Blimp-1 protein expression was assessed by immunoblot. Immunoblot with anti-GAPDH indicates equal protein loading. D, U937 cells were differentiated with PMA for 0, 2, 4, 8, and 24 h and analyzed as in C. Shown is a representative blot of three separate experiments.

The NLRP12 promoter contains a potential novel Blimp-1 NLRP12 and Blimp-1 protein expression are inversely binding site correlated during TLR stimulation and myeloid cell Our data thus far suggest that the down-regulation of NLRP12 differentiation mRNA after TLR stimulation involves transcriptional regulation. The identification of a potentially novel Blimp-1 binding site in the To identify potential factors involved in down-regulating NLRP12, NLRP12 promoter led us to question whether Blimp-1 is important we analyzed the first 622-bp sequence of the NLRP12 promoter for for the silencing of NLRP12 expression. We hypothesized that transcription factor-binding sites using TESS. We identified sev- NLRP12 expression might be down-regulated by an increased eral transcription factor binding sites including PU.1, which is in- level of Blimp-1 protein expression after TLR stimulation and cell volved in myeloid cell activation and differentiation (56) (Fig. 2A). differentiation. To assess this, we first examined the expression of Sequence analysis also revealed a potential binding site for NLRP12 and Blimp-1 proteins in the monocyte cell line U937 Blimp-1 encoded by the gene PRDM1 located at nucleotide posi- after stimulation with LPS for 0, 2, 4, 8 and 24 h (Fig. 2C, top tion Ϫ80 relative to the transcription start site (Fig. 2A) (57). All panel). We observed a rapid down-regulation of NLRP12 protein previously identified Blimp-1 binding sites contain the consensus expression with little NLRP12 protein detectable at 2 h after LPS A T T G sequence ( /C)AG( /C)GAAAG( /C)( /T) with the exception of stimulation. In contrast, Blimp-1 protein expression was increased A the NLR gene CIITA, which contains the sequence ( /C)AG at 2–4 h after TLR stimulation, and this increase continued at 8 T T G ( /C)GAAAT( /C)( /T) (49, 57, 58). Analysis of the putative and 24 h after TLR activation (Fig. 2C, bottom panel). Immuno- Blimp-1 binding site in NLRP12 suggests that this site contains the blots were probed with anti-GAPDH to indicate equal loading. A T T sequence ( /C)AG( /C)GAAAA( /C)(C), which is different from While the data suggest that NLRP12 and Blimp-1 protein expres- the previously identified consensus sequence (Fig. 2B). sion are inversely correlated, we initially observed a delay in The Journal of Immunology 2953

Blimp-1 protein induction at the 2 h time point. Our data suggest that in addition to Blimp-1, it is possible that other transcription factors are involved in the LPS-mediated down-regulation of NLRP12. In addition to the Blimp-1 binding site in the NLRP12 promoter, we observed several binding sites for PU.1. PU.1 is a positive regulator of gene transcription and is expressed in monocyte cells (59). More importantly, PU.1 is down-regulated by LPS (60), which may also attribute to the rapid reduction in NLRP12 protein after LPS stimulation. These data suggest that the inhibitory effect of TLR4 activation on NLRP12 protein expression is, in part, inversely correlated with its effect on Blimp-1 protein expression. Blimp-1 mediates cell differentiation in several cell types, including myeloid cells, and its expression is increased during myeloid-monocytic differentiation (45–48, 61–64). Thus, we examined if NLRP12 expression might also be inversely correlated with Blimp-1 during myeloid differentiation. To test this, the U937 cell line was treated with PMA for 0, 2, 4, 8, and

24 h to induce cell differentiation followed by analysis of Downloaded from NLRP12 and Blimp-1 protein expression (Fig. 2D). NLRP12 protein expression was down-regulated beginning at 2 h after PMA-induced differentiation, and expression disappeared by 4 h (Fig. 2D, top panel). Conversely, the induction of Blimp-1 protein expression after PMA stimulation began 4 h after PMA FIGURE 3. Blimp-1 binds to the putative Blimp-1 binding site in the addition (Fig. 2D, middle panel). Immunoblots were probed http://www.jimmunol.org/ human NLRP12 promoter and is regulated by PMA and TLR stimulation. with anti-GAPDH to indicate equal loading. These results show A, EMSA analysis of Blimp-1 DNA binding to the novel site in the that the decrease of NLRP12 during differentiation correlates NLRP12 promoter. Nuclear extracts were obtained from HEK293T cells with an increase in Blimp-1 protein expression. These data led transfected with a pcDNA control or a Flag-Blimp-1 expression vector. us to hypothesize that the induction of Blimp-1 expression dur- EMSA was performed using the Blimp-1 binding site in the NLRP12 pro- ing TLR stimulation and cell differentiation might be one mech- moter. Binding was competed with increasing concentrations of cold probe anism by which NLRP12 expression is down-regulated. to indicate specificity. An anti-Flag Ab was used to supershift the protein- DNA complexes to indicate specificity. A nonspecific anti-p65 Ab was Blimp-1 binds to the NLRP12 promoter after TLR stimulation used as a control. A representative of three separate experiments is shown. and differentiation B, Nuclear extracts from U937 cells stimulated with PMA and LPS for 24 h by guest on September 29, 2021 were used in a DAPA analysis of Blimp-1 binding to the NLRP12 pro- To address the potential role for Blimp-1 in regulating NLRP12 moter. DAPAs were performed by incubating U937 nuclear extracts with gene expression, we assessed if Blimp-1 could bind to the NLRP12 an immobilized, biotinylated double-stranded oligo corresponding to the promoter. EMSA was performed using a 30-bp oligonucleotide potential Blimp-1 binding site in the NLRP12 promoter. Bound protein containing the putative Blimp-1 binding sequence 5Ј-AAGT complexes were washed, eluted with sample buffer, and subjected to SDS- GAAAATC-3Ј in the NLRP12 promoter. Nuclear extracts were PAGE and Western blot analysis with a Blimp-1-specific Ab. A represen- prepared from HEK293T cells transfected with a pcDNA3 control tative DAPA of two separate experiments is shown. vector or Flag-tagged Blimp-1. EMSA revealed a DNA-protein complex in the lane containing Flag-Blimp-1 but not in the pcDNA3 vector control lane (Fig. 3A, left panel, lanes 3 and 2, respectively). This complex was competed by increasing amounts myeloid cells can bind to the NLRP12 promoter, and that this of cold competitive probe indicating specificity (Fig. 3A, left panel, binding is increased after TLR activation by LPS or cell differen- lanes 4 and 5). Addition of a Flag Ab to the binding reaction tiation with PMA. resulted in a slower migrating band, indicating the presence of Flag-Blimp-1 in the DNA-protein complex (Fig. 3A, right panel, NLRP12 expression is down-regulated by Blimp-1 lane 1). This complex was not shifted by a control anti-p65 Ab, The observation that Blimp-1 binds to a novel site on the NLRP12 indicating specificity of Ab binding (Fig. 3A, right panel, lane 2). promoter in vitro supports the idea that Blimp-1 mediates the re- These data show that Blimp-1 can bind to the novel Blimp-1 bind- pression of NLRP12 expression in vivo. To test this, we generated ing site found on the NLRP12 promoter. a luciferase reporter vector driven by 1412 bp of the NLRP12 We next used DAPA to test the binding of Blimp-1 to the promoter (NLRP12-luc) as well as a NLRP12-luc vector in which NLRP12 promoter to assess if increased binding could be observed the Blimp-1 binding site was mutated (mutNLRP12-luc) (Fig. 4A). after TLR stimulation or cell differentiation. We stimulated the The pGL3-control vector served as a positive control for transfec- U937 monocyte cell line with LPS or differentiated them with tion efficiency. The U937 cell line was transfected with NLRP12- PMA for 24 h and performed DAPA analysis (Fig. 3B). Nuclear luc, mutNLRP12-luc, or pGL3-control luciferase reporter vectors, extracts from control unstimulated U937 cells and LPS-stimulated and either co-transfected with Flag-Blimp-1 or stimulated with or PMA-treated U937 cells were incubated with double-stranded LPS (Fig. 4B). We observed a similar decrease in luciferase ac- biotinylated oligonucleotides containing the putative Blimp-1 tivity in cells containing the NLRP12-luc reporter vector when binding site in the NLRP12 promoter. Bound proteins were eluted stimulated with LPS or when cotransfected with Flag-Blimp-1. followed by immunoblotting with an Ab to Blimp-1. A dramatic This is comparable to the extent that Blimp-1 reduces the pIV increase in Blimp-1 signal was observed after cells were stimu- promoter of CIITA and to which Bach2 represses the Blimp-1/ lated with PMA or LPS. These results indicate that Blimp-1 in PRDM1 gene in promoter reporter assays (65, 66). Neither LPS nor 2954 Blimp-1 DOWN-REGULATES NLRP12 Downloaded from http://www.jimmunol.org/

FIGURE 4. The NLRP12 promoter is down-regulated by LPS stimulation and Blimp-1 expression in human monocyte cells. A, Schematic representation of luciferase reporter constructs generated to assess NLRP12 promoter luciferase activity. For promoter analysis, 1412 bp of the NLRP12 promoter was inserted into pGL3-luciferase to drive luciferase reporter expression (NLRP12-luc). A mutant NLRP12 promoter luciferase reporter was generated by mutating 4 bp located in the putative Blimp-1 binding site (mutNLRP12-luc). Deletions of the NLRP12 promoter were also cloned into pGL4.10-luciferase vector including a 399-bp fragment (pGL4-399), an 852-bp fragment (pGL4-852), and the full-length 1412-bp fragment (pGL4-1412). B, The U937 human monocyte cell line was transfected with the pGL3-control vector, Mon-1 luc, or mutMon-1 luc. After transfection, cells were either left unstimulated, by guest on September 29, 2021 stimulated with LPS, or transfected with a Flag-tagged Blimp-1 expression vector. Twenty-four hours later, cells were lysed and luciferase levels were determined as described in Materials and Methods. Error bars represent the SEM of four separate experiments. C, The U937 human monocyte cell line was transfected with the pGL3-control vector, NLRP12-luc, pGL4.10-luciferase vector, pGL4-399, pGL4-852, and pGL4-1412. After transfection, cells were either left unstimulated, stimulated with LPS, or transfected with a Flag-tagged Blimp-1 expression vector. Twenty-four hours later, cells were lysed and luciferase levels were determined as described in Materials and Methods. Error bars represent the SEM of three separate experiments.

Blimp-1 affected the mutNLRP12-luc vector that contained a mu- in luciferase activity in cells containing all of the NLRP12 reporter tated Blimp-1 site, indicating that the Blimp-1 binding site in the vectors when stimulated with LPS or when cotransfected with NLRP12 promoter is required for LPS or Blimp-1-mediated down- Flag-Blimp-1. A Student’s t test was used to determine signifi- regulation of NLRP12. As a control, cells were transfected with a cance where indicated. Taken together, these data suggest that the pGL3-control vector, which contains SV40 promoter-driven lucif- NLRP12 promoter is regulated by Blimp-1, and that a functional erase activity. When stimulated with LPS, pGL3-control cells had Blimp-1 binding site is necessary for LPS- or Blimp-1-mediated a higher level of luciferase reporter activity, supporting previous down-regulation of NLRP12 promoter. data that SV40 promoter activity is up-regulated by LPS stimulation (67). When cotransfected with Blimp-1, we did not observe a difference in pGL3-control reporter activity relative to control TLR stimulation induces Blimp-1 binding to the NLRP12 cells. promoter in vivo To confirm these results, we also cloned shorter regions of the Our observations suggested that Blimp-1 binds the NLRP12 pro- NLRP12 promoter and the full-length 1412-bp fragment into the moter in vitro as assessed by EMSA and DAPA analysis, and that pGL4.10 luciferase vector. The pGL4.10 luciferase vector was Blimp-1 can repress an NLRP12 promoter luciferase reporter con- chosen due to the improved levels of signal-to-noise ratio as com- struct. To determine whether Blimp-1 binds to the NLRP12 pro- pared with the older pGL3 luciferase version (Fig. 4A). The U937 moter in vivo in an LPS-induced manner, we performed ChIP as- cell line was transfected with NLRP12-luc, pGL3-control, says. U937 cells were stimulated with LPS for 8 h followed by pGL4.10 vector, pGL4 containing 399 bp of the NLRP12 promoter immunoprecipitation of protein-DNA complexes with anti-IgG or (pGL4-399), pGL4 containing 852 bp of the NLRP12 promoter with anti-Blimp-1. PCR using NLRP12 promoter-specific primers (pGL4-852), and pGL4 containing 1412 bp of the NLRP12 pro- that result in a 300-bp product surrounding the Blimp-1 site was moter (pGL4-1412) luciferase reporter vectors. Cells were either performed on input DNA, a 1/10 dilution of input DNA, and ChIP cotransfected with Flag-Blimp-1 or stimulated with LPS (Fig. 4C). was performed with anti-IgG or anti-Blimp-1 Abs followed by Consistent with our previous observations, we observed a decrease visualization by gel electrophoresis (Fig. 5A). Samples precipitated The Journal of Immunology 2955

cells for 0, 4, and 8 h followed by ChIP analysis with anti-IgG or anti-Blimp-1 Abs as described above (Fig. 5B). Blimp-1 association with the endogenous NLRP12 promoter increased by 2-fold 4 h after LPS stimulation, and 6-fold by 8 h. Primers speciﬁc for the NQO1 (NAD(P)H:quinone oxidoreductase) promoter, which is not regulated by Blimp-1, were used as a negative control (Fig. 5B). These data suggest that Blimp-1 associates with the NLRP12 promoter in a LPS-induced manner. Transcriptional silencing is often associated with decreased histone acetylation. Blimp-1 recruits the G9a methyltransferase and confers transcriptional silencing by inducing histone H3 acetylation and methylation (42). To assess if the binding of Blimp-1 to the NLRP12 promoter is correlated with a decrease in H3 acetylation, we stimulated U937 cells for 0, 4, and 8 h followed by ChIP with anti-IgG or anti-acetylated histone 3 Abs. We observed that acetylated histone 3 levels were reduced by 4-fold at the 4 and 8 h time points, suggesting that the NLRP12 promoter is being tran- scriptionally silenced (Fig. 5C). These data show that LPS reduced

transcriptional activity of the NLRP12 promoter is characterized Downloaded from by decreased histone acetylation and increased association of Blimp-1. Endogenous Nlrp12 gene expression is regulated by Blimp-1 in primary cells

The observation that NLRP12 and Blimp-1 protein expression is http://www.jimmunol.org/ inversely correlated in human myeloid cells during LPS stimulation and differentiation led us to examine the expression of these two genes in primary myeloid cells from mice. However, we have not observed decreased Nlrp12 expression upon LPS stimulation of mouse bone marrow cells, likely reflecting species variation in the regulation of this gene. Thus, we concentrated on Nlrp12 expression during myeloid differentiation. In vitro differentiation of murine bone marrow with GM-CSF induces the differentiation of hematopoietic progenitors into more mature dendritic cells and by guest on September 29, 2021 macrophages. To determine whether Nlrp12 and Blimp-1 expression is altered during differentiation of mouse bone marrow cells, we isolated bone marrow from C57BL/6 mice and differentiated FIGURE 5. Blimp-1 is recruited to the NLRP12 promoter after TLR these cells in vitro for a period of 5 days with GM-CSF. We ob- stimulation in vivo. A, Human U937 cells were stimulated with LPS for 8 h served a decrease in Nlrp12 expression over the GM-CSF differ- followed by ChIP using a control IgG Ab, an Ab to acetylated histone 3, entiation period as assessed by qPCR, but not by LPS stimulation and an Ab to Blimp-1. DNA was eluted from the ChIP and subjected to of murine cells as expected (Fig. 6A). Conversely, Blimp-1 ex- PCR with primers specific for the NLRP12 promoter. PCR products were analyzed on an agarose gel. Lanes include PCR product obtained from pression was increased during the differentiation period. Thus, input DNA, a 1/10 dilution of input DNA, immunoprecipitation with an Nlrp12 and Blimp-1 expression are inversely correlated during dif- IgG control Ab, immunoprecipitation with an anti-Blimp-1 Ab, and water ferentiation of murine bone marrow cells with GM-CSF in vitro. as a PCR control. Data are representative of three separate experiments. B, Next, we determined whether Blimp-1 mediates the transcrip- ChIP was performed to confirm the interaction of Blimp-1 with the tional repression of Nlrp12 during murine bone marrow differen- NLRP12 promoter in vivo. The human monocyte cell line U937 was stim- tiation. We obtained cells from Rosa26ERCre/ϩprdm1flox/flox Cre- ulated with LPS for 1, 4, and 8 h. Chromatin-bound Blimp-1 complexes lox mice made from the Rosa26 ES cell line, which ubiquitously were immunoprecipitated using an anti-Blimp-1 Ab followed by analysis expresses lacZ in adult mouse tissues as described previously (68). of complexed chromatin by qPCR using primers specific for the NLRP12 For our studies, Blimp-1-null bone marrow cells were obtained promoter. qPCR using primers specific for the NQO1 promoter were used ϩ from Rosa26ERCre/ prdm1flox/flox mice following treatment of the as a negative control. qPCR values have been normalized to input. Pre- cipitation with an unrelated Ab was used as negative control. Data are mice with tamoxifen to activate the Cre recombinase, resulting in representative of three separate experiments. C, ChIP was performed to deletion of the Blimp-1 gene (68). Isolated Blimp-1-deficient bone measure acetylated-histone 3 (H3) levels with the NLRP12 promoter in marrow cells were differentiated in vitro with GM-CSF for the in- vivo. The human monocyte cell line U937 was stimulated with LPS for 1, dicated time points (Fig. 6B). GM-CSF caused a steady decrease in 4, and 8 h. Chromatin-bound acetylated-H3 complexes were immunopre- Nlrp12 expression over a period of 5 days as shown in the control cipitated using an anti-acetylated-H3 Ab followed by analysis of com- Blimp-1-expressing cells from Rosa26ϩ/ϩprdm1flox/flox mice, al- plexed chromatin by qPCR using primers specific for the NLRP12 pro- though the data did not reach statistical significance. From days moter. qPCR values have been normalized to input. Data represent an 1–4 of GM-CSF treatment, Nlrp12 was reduced in the Blimp-1- average of four experiments. deficient Rosa26ERCre/ϩprdm1flox/flox cells but to a much lesser extent relative to the control Blimp-1-expressing Rosa26ϩ/ϩ with the anti-Blimp-1 Ab show that endogenous Blimp-1 is re- prdm1flox/flox cells. By day 5, expression was reduced in both cell cruited to the NLRP12 promoter in vivo after TLR stimulation. To types regardless of the presence of Blimp-1 (Fig. 6B). It may by assess the kinetics of Blimp-1 binding, we then stimulated U937 possible that additional transcription factors including PU.1 are 2956 Blimp-1 DOWN-REGULATES NLRP12

FIGURE 6. Reduced expression of NLRP12 during murine bone marrow differentiation is regulated by Blimp-1. A, Murine bone marrow cells were isolated from C57BL/6 mice and differentiated in vitro with GM-CSF or stimulated with LPS for 3 h. Blimp-1 and NLRP12 expression were assessed by qPCR analysis of RNA isolated at the indicated time points. Data are representative of three separate experiments. B, Murine bone marrow cells were isolated from control ERϩ/ϩprdm1flox/flox mice or from ERCreϩprdm1flox/flox (48) mice in which Blimp-1 expression is knocked out under the control of the estrogen receptor Downloaded from promoter. Bone marrow cells were differentiated in vitro with GM-CSF. NLRP12 expression was assessed by qPCR analysis of RNA isolated at the indicated time points. Data are an average of at least three separate experiments, and error bars were calculated using SEM values. http://www.jimmunol.org/

also involved. The data indicate that Blimp-1 reduces Nlrp12 dur- “turn off” downstream activation by associating with TLR pathway by guest on September 29, 2021 ing the early points of GM-CSF stimulation but its expression is signaling proteins to once expressed. Based on our present under- reduced by additional mechanism(s) after prolonged GM-CSF standing of the function of NLRP12, we hypothesize that NLRP12 stimulation. Thus, inhibition of Nlrp12 expression observed during is present before immune stimulation to maintain a quiescent phe- differentiation of murine bone marrow cells is mediated in part by notype by inhibiting inappropriate TLR activation in the absence Blimp-1. of appropriate signals. We further hypothesize that during states of infection NLRP12 initially functions to modulate overzealous im- Discussion mune responses by associating with and regulating the activity of Based on in vitro analysis as well as the analysis of patients with TLR signaling proteins, including IRAK-1 and NIK (37, 38). Since NLRP12 splicing or nonsense mutations, this gene has emerged as we have observed that NLRP12 regulates both TLR and TNFR a negative regulator of inflammatory gene induction. NLRP12 pathways, we hypothesize that at later time points after TLR ac- modulates TLR inflammatory gene induction by its inhibition of tivation or during states of myeloid differentiation NLRP12 ex- IRAK-1 hyperphosphorylation (37) and NIK activation (38). The pression is decreased to allow a more complete immune response negative regulatory functions of NLRP12 have been shown to re- to occur. The hypothesis that NLRP12 initially functions to mod- quire ATP binding (69). In human myeloid cells, NLRP12 expres- ulate overzealous immune responses is supported by the recent sion is down-regulated both by cellular activation through TLR observation that humans with mutations in NLRP12 are predis- stimulation as well as developmentally after exposure of myeloid posed to the autoimmune syndrome known as HPF (39). Future cells to TNF-␣ and IFN-␥ (36). While NLRP12 is expressed in characterization of the function of NLRP12 will likely lead to a monocytes, it is not expressed in differentiated macrophages, sug- greater understanding of the role of NLRP12 down-regulation after gesting that the regulation of NLRP12 is under developmental control (35–37). Thus, the differentiation of monocytes into macro- TLR activation and myeloid differentiation. phages and the inflammatory response after TLR stimulation This report shows that Blimp-1 represses the NLRP12 promoter involve the repression of NLRP12. in transformed and primary myeloid-monocytic cells. This is me- The observation that NLRP12 expression is down-regulated af- diated by the binding of Blimp-1 to the NLRP12 promoter in intact ter TLR-mediated activation of human myeloid cells and by my- cells, resulting in a correlative loss of histone acetylation. The eloid differentiation of monocytes to macrophages is unusual for regulation of NLRP12 gene expression by Blimp-1 is most con- most innate immune genes. More often, proteins that function as vincingly shown using a Blimp-1 Cre-knockout mouse where the negative regulators of TLR signaling are induced after activation. down-regulation of NLRP12 during GM-CSF-dependent differen- Ϫ Ϫ An example of TLR-induced expression of negative regulators in- tiation of bone marrow progenitors is lessened in Blimp-1 / cludes the induction of IRAK-M (70) as well as the induction of cells. These data indicate that Blimp-1 reduces NLRP12 expression A20 (71), which are both induced 2–3 h after LPS exposure, and during cell differentiation and TLR signaling. The Journal of Immunology 2957

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