PILRα Negatively Regulates Mouse Inflammatory Arthritis Yonglian Sun, Patrick Caplazi, Juan Zhang, Anita Mazloom, Sarah Kummerfeld, Gabriel Quinones, Kate Senger, Justin This information is current as Lesch, Ivan Peng, Andrew Sebrell, Wilman Luk, Yanmei of September 24, 2021. Lu, Zhonghua Lin, Kai Barck, Judy Young, Mariela Del Rio, Sophie Lehar, Vida Asghari, WeiYu Lin, Sanjeev Mariathasan, Jason DeVoss, Shahram Misaghi, Mercedesz Balazs, Tao Sai, Benjamin Haley, Philip E. Hass, Min Xu,

Wenjun Ouyang, Flavius Martin, Wyne P. Lee and Ali A. Downloaded from Zarrin J Immunol published online 16 June 2014 http://www.jimmunol.org/content/early/2014/06/16/jimmun ol.1400045 http://www.jimmunol.org/

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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 © 2014 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published June 16, 2014, doi:10.4049/jimmunol.1400045 The Journal of Immunology

PILRa Negatively Regulates Mouse Inflammatory Arthritis

Yonglian Sun,* Patrick Caplazi,† Juan Zhang,* Anita Mazloom,‡ Sarah Kummerfeld,x Gabriel Quinones,‡ Kate Senger,* Justin Lesch,* Ivan Peng,* Andrew Sebrell,{ Wilman Luk,‖ Yanmei Lu,‖ Zhonghua Lin,* Kai Barck,# Judy Young,‖ Mariela Del Rio,** Sophie Lehar,†† Vida Asghari,** WeiYu Lin,* Sanjeev Mariathasan,†† Jason DeVoss,* Shahram Misaghi,‡‡ Mercedesz Balazs,* Tao Sai,{ Benjamin Haley,‡ Philip E. Hass,‡ Min Xu,* Wenjun Ouyang,* Flavius Martin,* Wyne P. Lee,* and Ali A. Zarrin*

Paired Ig-like type 2 receptor (PILR)a inhibitory receptor and its counterpart PILRb activating receptor are coexpressed on myeloid cells. In this article, we report that PILRa, but not PILRb, is elevated in human rheumatoid arthritis synovial tissue and correlates with inflammatory cell infiltration. Pilra2/2 mice produce more pathogenic cytokines during inflammation and are Downloaded from prone to enhanced autoimmune arthritis. Correspondingly, engaging PILRa with anti-PILRa mAb ameliorates inflammation in mouse arthritis models and suppresses the production of proinflammatory cytokines. Our studies suggest that PILRa mediates an important inhibitory pathway that can dampen inflammatory responses. The Journal of Immunology, 2014, 193: 000–000.

mmune responses are modulated by a network of positive- ITAM-bearing DAP12 adaptor molecule to deliver activating si-

and negative-regulatory mechanisms. consist gnals (8). Human and mouse PILRa share only ∼40% homology http://www.jimmunol.org/ I of highly related activating and inhibitory receptors that (7), yet conserved residues mediate ligand interactions (9). are widely involved in the regulation of the immune system (1). PILRa and PILRb transcripts show similar tissue expression, Both inhibitory and activating receptors share high similarity with high levels in spleen, liver, and lung and lower levels in in their extracellular domain, whereas their intracellular signal- the small intestine (8). PILRa and PILRb are predominantly ing domains are divergent (2, 3). Paired Ig-like type 2 receptor expressed in cells of the myelomonocytic lineage, including (PILR)a belongs to the Ig superfamily. Its intracellular domain monocytes, macrophages, granulocytes, and monocyte-derived contains two ITIMs that recruit SHP-1 and SHP-2 to trigger an dendritic cells (DCs) (4, 8). Additionally, PILRb is expressed in inhibitory signaling cascade, resulting in reduced intracellular NK cells (8). It was shown that PILRa binds to mouse CD99 (8), calcium mobilization (4, 5). PILRa and its -linked activating PILR-associating neural (10), and HSV-1 glycoprotein B by guest on September 24, 2021 counterpart, PILRb, share highly similar extracellular domains, (11). Specific sialylated O-linked glycans on ligands are required suggesting that they may recognize the same ligands (5–7). PILRb for their binding to PILRa (6, 10, 12). We recently identified two has a truncated cytoplasmic domain and a charged amino acid more binding partners of PILRa, neural proliferation differentia- residue in its transmembrane region that associates with the tion and control-1 and collectin-12, and found that an evolution- arily conserved PILRa domain mediates its interaction with these diverse sialylated ligands (9). This suggests that a complex net- *Department of Immunology, Genentech, South San Francisco, CA 94080; work of ligands might modulate cellular functions via PILRa.It †Department of Pathology, Genentech, South San Francisco, CA 94080; was shown that PILRa binds to HSV-1 glycoprotein B and serves ‡Department of Protein Chemistry, Genentech, South San Francisco, CA 94080; x as a virus entry coreceptor (11, 13). Previous studies showed that Department of Bioinformatics and Computational Biology, Genentech, South San Francisco, CA 94080; {Department of Engineering, Genentech, South San PILRb-deficient and agonist anti-PILRa–treated mice show im- ‖ Francisco, CA 94080; Department of Biochemical and Cellular Pharmacology, Gen- proved clearance of infectious reagents and improved survival (14, entech, South San Francisco, CA 94080; #Department of Biomedical Imaging, Gen- entech, South San Francisco, CA 94080; **Department of Transgenic Technology, 15). A recent study showed that PILRa negatively regulates Genentech, South San Francisco, CA 94080; ††Department of Microbial Pathogen- neutrophil recruitment during TLR-mediated inflammatory responses ‡‡ esis, Genentech, South San Francisco, CA 94080; and Department of Early Stage (16). However, the role of PILRa or PILRb in autoimmunity is Cell Culture, Genentech, South San Francisco, CA 94080 largely unknown. Received for publication January 9, 2014. Accepted for publication May 19, 2014. Myeloid cells play a critical role in the pathophysiology of The microarray data presented in this article have been submitted to the National inflammation and autoimmunity. Rheumatoid arthritis (RA) is Center for Biotechnology Information’s Omnibus (http://www.ncbi. nlm.nih.gov/geo/query/acc.cgi?acc=GSE48780) under accession number GSE48780. a systemic, inflammatory, autoimmune disorder manifested by Address correspondence and reprint requests to Dr. Ali A. Zarrin, Genentech, 1 DNA chronic polyarthritis with synovial hyperplasia and joint destruc- Way, South San Francisco, CA 94080. E-mail address: [email protected] tion, resulting in pain, loss of joint function, and concomitant The online version of this article contains supplemental material. reduction of life quality (17, 18). Myeloid cells, including Abbreviations used in this article: BM, bone marrow; BMDC, BM-derived DC; monocytes/macrophages and neutrophils, play an important role BMDM, BM-derived monocyte; CAIA, collagen Ab–induced arthritis; CLEC4G, in various stages of arthritis development (19, 20). To investigate C-type lectin domain family 4, member G; DC, dendritic cell; ES, embryonic stem; h, human; JCBV, joint cortical bone volume; m, mouse; micro-CT, microcomputed the role of PILRa in myeloid cell–mediated immune responses tomography; MPO, myeloperoxidase; OA, osteoarthritis; PILR, paired Ig-like type 2 and its effects on autoimmune diseases, we studied its function receptor; PNBV, periosteal new bone volume; RA, rheumatoid arthritis; WT, wild- in myeloid-driven models of RA, including collagen Ab–induced type. arthritis (CAIA) and K/BxN serum–transfer arthritis, using PILRa- Copyright Ó 2014 by The American Association of Immunologists, Inc. 0022-1767/14/$16.00 deficient mice and PILRa-specific mAbs. Murine CAIA is in-

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1400045 2 PILRa REGULATES INFLAMMATION ducedbyi.v.injectionofmAbsagainst type II collagen, fol- mAbswasconfirmedbyELISAandflowcytometryanalysis.ThesemAbsdo lowedbyi.p.injectionofLPS.Thismodeliswidelyusedto not cross-react with mouse PILRb. Hamster anti-PILRa mAb was used for study the pathogenesis of autoimmune arthritis and to determine staining, and mouse anti-PILRa mAb was used for in vivo and in vitro functional studies. efficacy of therapeutics (21–24). Myeloid cells, FcgRs (22), and proinflammatory cytokines, especially TNF-a and IL-1b (21), are Gene expression analysis indispensable for the development and maintenance of arthritis in For gene expression analysis, total RNA was isolated using TRIzol reagent this model (23). The K/BxN serum–transfer arthritis model is (Invitrogen). Total RNA (50 ng) was subjected to RT-PCR using Access RT- induced by transferring K/BxN serum into normal mice; LPS is PCR System (Promega). Primers used for amplification were as follows: not needed in this model. The K/BxN serum–transfer arthritis b-actin, sense primer: 59-TACCTCATGAAGATCCTCA-39 and antisense model shares multiple features with human RA, including sym- primer: 59-TTCGTGGATGCCACAGGAC-39; PILRa, sense primer: 59- GACTCCTGAGCACTCCAGTGTCCCTAGACAG-39 and antisense primer metrical involvement of peripheral joints, pannus formation, sy- 59-AAGGTGACTCTAAGAGTGTCATCCAGACCA-39;andPILRb,sense novial hyperplasia, and bone and cartilage degradation (25). We primer: 59-GGAAATTCAGAAAGATACAACA-39 and antisense primer: 59- show that Pilra2/2 mice developed more severe arthritis than did CAGGTTCATCAGTGAAGGAT-39. For quantitative RT-PCR, total RNA wild-type (WT) mice in these two models, corroborating the in- (50 ng) was subjected to RT and real-time quantitative PCR assay using Access RT-PCR System (Promega), Perkin Elmer SYBR Green, and an ABI hibitory role of PILRa during inflammation. Accordingly, we 7500 instrument. Gene-specific unlabeled primers used were as follows: found that administration of an anti-PILRa mAb attenuated b-actin, sense primer: 59-TACCTCATGAAGATCCTCA-39 and antisense mouse inflammatory arthritis and reduced the production of sev- primer: 59-TTCGTGGATGCCACAGGAC-39)PILRb,senseprimer:59- eral proinflammatory cytokines, indicating that PILRa is part of CACCATGAGGAGCCCCTTCATCGTC-39 and antisense primer: 59- a critical inflammatory axis regulating myeloid cell function. TAGGACTATGACCAAAACTTTAGCCAGGAG-39. Downloaded from Cell extraction and Western blot analysis Materials and Methods Bone marrow (BM) cells and splenocytes were harvested, and RBCs were Mice lysed with ACK lysing buffer. Cells were washed twice with ice-cold PBS (pH 7.4) and then treated in Cell Extraction Buffer (Invitrogen) containing BALB/c mice were purchased from Charles River Laboratory. Pilra-defi- 1 mM PMSF and protease inhibitor mixture (Sigma). Lysates were incu- cient mice were generated by genOway and were maintained in-house.

bated at 4˚C for 30 min, with vortexing at 10-min intervals, and the in- http://www.jimmunol.org/ Mice were used at 6–10 wk of age. All animal experiments were per- soluble material was removed by centrifugation at 13,000 rpm for 10 min formed in accordance with institutional animal care and use committee– at 4˚C. Soluble extracts with Novex Tris-Glycine SDS sample buffer were approved protocols. separated by SDS-PAGE using 4–12% Bis-Tris gel and transferred to Generation of Pilra2/2 mice a PVDF/Filter Paper Sandwich (all from Invitrogen). Blots were blocked with 5% nonfat dried milk, 0.1% Tween-20 in TBS buffer and incubated The genomic region containing the murine PILRa locus was isolated by with hamster anti-mouse PILRa mAb, followed by HRP-conjugated anti- PCR from 129Sv BAC genomic DNA (genOway, Lyon, France). The hamster IgG (Jackson ImmunoResearch) for 1 h. were detected by 6050-bp 59 homology arm corresponds to nt 137843309–137837259 chemiluminescence with an Amersham ECL Plus Western blotting system cluster NC_000071.6 contig GRCm38.p2 in 5 of C57BL/6J. (GE Healthcare). The 1872-bp 39 homology arm corresponds to nt 137830963–137829091 of the same region. This strategy allows for deletion of a 6.3-kb region Immunohistochemistry by guest on September 24, 2021 a comprising exons 1 and 2 of PILR (containing ATG, Ig V-set domain, Immunohistochemistry for PILRa was performed on formalin-fixed, and extracellular domain). Neomycin-resistant clones were first screened paraffin-embedded tissue using in-house–generated Armenian hamster 9 9 by PCR using primers 5 -ATGCTCCAGACTGCCTTGGGAAAAG-3 anti-mPILRa mAb and an automated system (VENTANA DISCOVERY 9 (neocassette) and antisense 5 -CACTTCACAGTCTAGTCACTTCCGC- XT, Ventana Medical Systems, Tucson, AZ). Briefly, 4-mm sections were 9 9 ACC-3 (outside 3 homology arm). Final clones were confirmed using deparaffinized, rehydrated, and subjected to Ag retrieval (Ventana Protease 9 9 9 primers 5 -GCACCATGTCCTGAGGAAGAGTAGTTTGA-3 (within 5 2). Primary Ab (5 mg/ml) was incubated at 37˚C for 60 min. Binding was 9 9 homology arm) and 5 -CACTTCACAGTCTAGTCACTTCCGCACC-3 visualized using secondary anti-hamster IgG, Ventana Rabbit OmniMap, 9 (within 3 homology arm), which generates a 7.0-kb fragment in WT and Ventana DAB reagents (Ventana Medical Systems). Immunohisto- embryonic stem (ES) cells or a 3.8-kb fragment in neo-containing targeted chemistry for myeloperoxidase (MPO) and F4/80 was performed on ES cells. The expected neo-excised fragment of 2.1 kb was cloned and formalin-fixed, acid-decalcified, paraffin-embedded sections of murine sequenced to confirm the desired recombination event for all ES clones. arthritic paws to visualize infiltration by neutrophils or macrophages, Gene targeting was further confirmed by Southern blot using a neomycin respectively. For MPO, a rabbit polyclonal Ab A95-1 (LabVision/ 9 internal probe (SacI digest generating an 8.6-kb fragment) and a 3 ex- Neomarker, Fremont, CA) was used on an automated platform (Ven- 9 ternal probe (SphI digest changing 11.0 kb to 6.0 kb). The 3 probe con- tana) using Ventana Rabbit OmniMap and DAB for visualization. For F4/ 9 tains a 450-bp fragment between the 3 end of the targeting arm and the 80, a commercial rat Ab (Cl:A3-1; AbD Serotec, Raleigh, NC) was used SphI site. A total of 6 of 646 clones carried correct integration. Two in- on an automated platform (Leica Bond) and visualized with DAB using an dependent clones were microinjected into C57BL/6J-Tyrc-2J/J blastocysts ABC Peroxidase protocol. to generate chimeras, which were bred to C57BL/6N mice (Charles River) to generate PILRa-knockout mice. Subsequently, the neomycin cassette Flow cytometry was removed by breeding PILRa-knockout mice carrying the neomycin cassette with C57BL/6N Rosa-26-cre knockin mice (Taconic). The fol- Single-cell suspensions were prepared from BM and peritoneal lavage fluid. lowing primers were used to confirm the cre recombination and genotype: After RBC lysis, cells were incubated with rat anti-mouse CD16/CD32 forward primer-1: 59-TGCAAACCACAGTTGGGTTGGCAACGGCTG-39 (Mouse BD Fc Block) and stained with in-house–generated hamster anti- (located in exon 2, which is deleted in the knockout) and reverse primer: mouse PILRa mAb, followed by allophycocyanin-conjugated anti-hamster 59-GGAGGAGGCCAGAAGCCAACCTTTGCTATC-39 (within 39 target- IgG (R&D Systems). BM cells were further stained with PE-conjugated ing arm, which generates a 470-bp fragment in WT mice) or forward Ly6G and FITC-conjugated anti-CD11b. Peritoneal cells were stained with primer-2: 59-ATGACAGTTTGTGTCTGGATCAGAAC-39 (upstream of PE-conjugated Gr-1 and FITC-conjugated anti-CD11b or FITC-conjugated the loxp site) and the same reverse primer, which generates a 600-bp CD117 and PE-conjugated FcεRI (all from BD Pharmingen). Cell acquisi- product from the knockout allele. tion was performed on a FACSCalibur (Becton Dickinson, Mountain View, CA), and data were analyzed with FlowJo software. Generation of mAbs Induction of CAIA Anti-PILRa mAbs were generated from Armenian hamsters and Pilra2/2 mice using immunogenic fusion proteins consisting of the extracellular Arthritis was induced in 7–8-wk-old (age-matched for a given study) fe- domain of the mouse Pilra gene (GenBank accession number: NM_153510), male C57BL/6N or BALB/c mice with an arthritogenic anti–type II col- including amino acid positions Met 1 to Val 197, as previously described lagen mAb mixture purchased from Chondrex. Pilra2/2 and WT littermate (9). Mouse anti-PILRa mAbs are IgG1 isotype. The specificity of anti-PILRa mice received 4 mg a mixture of anti–type II collagen mAbs i.v. in 400 ml The Journal of Immunology 3

PBS on day 0, followed by 50 mg LPS i.p. in 100 ml PBS on day 3. To test Microcomputed tomography imaging the effect of anti-PILRa mAb on the development of CAIA, BALB/c mice received 2 mg Arthrogen-CIA Ab mixture i.v. in 200 ml PBS on day 0, Microcomputed tomography (micro-CT) imaging was performed using followed by 50 mg LPS i.p. in 100 ml PBS on day 3. Mice were treated a previously described micro-CT and automated analysis technique (26) to with 100 mg mouse anti-PILRa mAb or anti-ragweed mIgG1 isotype quantify bone destruction in the arthritis mouse model. Briefly, the paws control i.p. daily in 100 ml sterile saline, starting on day 21. For K/BxN were scanned ex vivo on a micro-CT 40 scanner (Scanco Medical, € serum–transfer arthritis model, BALB/c mice were given 20 ml arthrito- Bruttisellen, Switzerland). The analysis algorithm automatically locates genic K/BxN serum on days 0 and 2 by i.v. injection and were treated five metatarsophalangeal joints and three metacarpophalangeal joints and with 100 mg mouse anti-PILRa mAb or anti-ragweed mIgG1 isotype segments the cortical bone and periosteal new bone. Joint cortical bone control i.p. daily in 100 ml sterile saline starting on day 21. Swelling of the volume (JCBV) is sensitive to cortical bone erosion at the joints, and paws was monitored for 11–12 d. Each paw was assigned a score based on periosteal new bone volume (PNBV) estimates the amount of periosteal the following scale: 0 = no evidence of erythema and swelling; 1 = erythema new bone formation that results from a repair response to the bone erosion. and mild swelling confined to the midfoot (tarsal) or ankle; 2 = erythema and The analysis technique was modified in such way that JCBV, as well as the mild swelling extending from the ankle to the midfoot; 3 = erythema and PNBV estimate, was limited to the joints. moderate swelling extending from the ankle to the metatarsal joints; and 4 = Measurement of cytokine concentration in arthritic hind paws erythema and severe swelling encompass the ankle, foot, and digits. The clinical score of each mouse is the sum of the four paw scores. Hind footpads were obtained from mice by cutting at the borderline of fur growth. The footpads were hammer smashed and shaken at 20 Hz for Histopathological assessment 30–60 min with 5-mm steel beads at 4˚C in ice-cold cell lysis buffer Paws were removed, fixed in formalin, decalcified, and processed to ( Technology) containing Protease Inhibitor Cocktail Set I paraffin sections stained with H&E. Sagittal sections were examined by (Calbiochem). Homogenized tissue was kept on ice for 30 min, vor- texed occasionally, and then spun down at high speed for 15 min at 4˚C. lightmicroscopyandscoredonarbitraryscalesrangingfrom0to5for 2 Downloaded from features of arthritis, including number of affected joints, extent of in- Supernatants were collected, and all samples were frozen at 80˚C and flammation, extent of soft tissue proliferation including pannus, cartilage thawed to room temperature before analysis. Cytokine levels were ana- injury, and bone remodeling. Immunoreactivity for MPO or F4/80 was lyzed by ELISA (R&D System). The concentration of total protein in the evaluated on arthritic paws by microscopic examination of affected sy- supernatants was measured using a Pierce BCA kit and was normalized novial structures (joints, tendon sheaths). Intensity of infiltration with against the concentration of BSA. The concentration of cytokines was immunoreactive cells was estimated using an arbitrary scoring scheme expressed as pg/mg of protein. ranging from 0 to 3, according to the following criteria: 0 = none In vitro stimulation of BM-derived DCs detected near or in synovial structures; 1 = few, scattered positive cells http://www.jimmunol.org/ near or in synovial structures; 2 = frequent disseminated or loosely BM-derived DCs (BMDCs) were generated as described elsewhere (27). In clustered positive cells near or in synovial structures; and 3 = locally brief, femurs of mice were removed, and BM was flushed out with PBS extensive infiltration with numerous positive cells near or in synovial containing 10% FCS. RBCs were lysed with ACK lysing buffer. Cells were structures. cultivated in bacterial culture dishes for 9–10 d at 37˚C in a humidified by guest on September 24, 2021

FIGURE 1. Pilra2/2 mice generation and char- acterization. (A) Schematic diagram of PILRa-tar- geting construct. Exons 1 and 2 were replaced with loxp sites (triangles) flanked by the neomycin re- sistance gene (neo). The neomycin cassette was deleted by crossing the mice with the Rosa-26-cre knockin line. (B) Genomic DNA from WT ES cells or targeted ES cells were digested with SacI and probed with a neomycin internal probe (left panel). All independent clones detected a single band of an 8.6-kb fragment corresponding to a single integra- tion event at the 59 homology arm. SphI genomic DNA digest was used to confirm expected homol- ogous recombination at the 39 end using a genomic probe located between the 39 end of the targeting arm and the SphI site, which generated a shorter fragment of 6.0 kb compared with 11.0 kb in WT ES cells (right panel). (C) PILRa and PILRb ex- pression in spleen and BM of WT and Pilra2/2 (KO) mice by RT-PCR (n = 3). (D) PILRb ex- pression in spleen and BM of WT and Pilra2/2 (KO) mice by real-time PCR. (E) PILRa expression in spleen and BM of WT, Pilra+/2 (HET), and Pilra2/2 (KO) mice by Western blot. Data are representative of two or three experiments. 4 PILRa REGULATES INFLAMMATION

atmosphere containing 5% CO2 using RPMI 1640 supplemented with 10% Clara, CA). Samples were hybridized to GeneChip U133 heat-inactivated FCS, 2 mM L-glutamine, 100 U/ml penicillin, 100 mg/ml Plus 2.0 Arrays (Affymetrix). Arrays were washed and stained in the Af- streptomycin, 50 mM 2-ME (Sigma; Taufkirchen, Germany), and 20 ng/ml fymetrix Fluidics station and scanned on a GeneChip scanner 3000. Ex- recombinant mouse (m)GM-CSF (R&D Systems). At days 9–10, the pression signals were obtained using the Affymetrix GeneChip operating nonadherent cells were harvested and stimulated with mIgG (60 mg/ml; system and analysis software. Data have been deposited in the National Jackson ImmunoResearch Laboratories) plus various concentrations of Center for Biotechnology Information’s Gene Expression Omnibus under mouse anti-mouse PILRa mAb coated on Protein G Agarose beads (Roche) accession no. GSE48780 (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi? for 18 h. Cytokine production in supernatants was detected by ELISA acc=GSE48780). (R&D) or Luminex (Bio-Rad). Computational analysis. Computational analysis was carried out using the R a b programming language (29) and libraries from the open-source Bioconductor PILR and PILR expression in synovial tissues of RA and project (30). Quality assessment of microarray data was conducted using the osteoarthritis patients Bioconductor affy package (31), and the expression data were normalized Patients and synovial tissues. Synovial tissues were obtained from RA using the robust multiarray average method (32, 33). PILRa and PILRb are subjects undergoing arthroplasty and/or synovectomy of affected joints each represented by two probes on the Affymetrix U133Plus chips. (University of Michigan, two sequential cohorts). RA was diagnosed based To compare the expression levels of PILRa and PILRb in RA and upon the 1987 American College of Rheumatology criteria (28). Patients were osteoarthritis (OA) samples, we used a linear model with terms for diag- treated using the standard of care for RA (nonsteroidal anti-inflammatory nosis (OA or RA) and inflammation status (inflamed or noninflamed) based drugs and disease-modifying antirheumatic drugs), and some patients also on histology. Conceptually, this can be thought of as a t test comparing OA were treated with biologics (adalimumab, etanercept, infliximab, anakinra, and RA expression with correction for inflammation status. The limma and rituximab). Patients were diagnosed with RA $3 y before surgery, and Bioconductor package was used for the computation (34). 70% of patients for whom data were available were positive for rheumatoid factor. All procedures to collect human specimens were performed under PILRa ligand screen Downloaded from a protocol approved by the University of Michigan Institutional Review Recombinant human PILRa-Fc was screened for interacting partners using Board. Excised tissues were immediately snap-frozen in liquid nitrogen and 2 the Genentech human full-length cDNA expression library. COS-7 cells stored at 80˚C. Each tissue was used for both histology and RNA ex- were transfected with 6 ng/ml individual cDNA expression vectors and traction. For cryosectioning, samples were brought briefly to 220˚C, sec- 2 FuGene 6 (Roche) in a 384-well format. Forty-eight hours after transfec- tioned, and immediately returned to 80˚C to maintain RNA integrity. All tion, COS-7 cells were exposed to 10 ng/ml PILRa-Fc, followed by fix- tissues used for downstream studies were prospectively randomized during ation in 4% formaldehyde. Protein–protein interactions were detected upon processing and sectioning, prior to expression analysis, to minimize technical binding of an Alexa Fluor 488–conjugated anti-mIgG2a secondary Ab batch effects in the data. (Invitrogen) to the cell surface using an MD Isocyte plate scanner or, in http://www.jimmunol.org/ RNA isolation. Frozen samples were weighed and homogenized in RLT follow-up experiments, using a GE IN Cell 2000 Analyzer. (QIAGEN, Valencia, CA) + 2-ME (10 ml/ml) at a concentration of 100 mg/ To confirm the binding of screening hit to PILRa, HEK 293T cells ml. RNA was isolated using an RNeasy Mini Kit (QIAGEN) with on- were transfected with C-type lectin domain family 4 (CLEC4G) cDNA, column DNase digestion. and transfectants were stained with human (h)PILRa-Fc/mPILRa-Fc, Microarray hybridization. The protocols for preparation of cRNA and for hPILRaR126A-Fc/mPILRaR133A-Fc, followed by FITC-conjugated anti- array hybridization were followed as recommended by Affymetrix (Santa mIgG2a. Cell acquisition was performed on a FACSCalibur (Becton by guest on September 24, 2021

FIGURE 2. Expression of PILRa in various tis- sues and cells. (A) Immunohistochemistry of nor- mal murine tissues. Tissue sections from WT mouse spleen, liver, lung, and BM were stained with anti-PILRa Ab and compared with control tissues from Pilra2/2 mice. Spleen red pulp and tissue-resident macrophages, such as Kupffer cells in liver (inset) or exfoliated alveolar macrophages (inset) in lung, express PILRa. In BM, most PILRa+ cells are identified as granulocytes or their precursors. Scale bars, spleen = 500 mm; liver and lung = 50 mm; BM = 20 mm. (B) PILRa is expressed on murine granulocytes and monocytes from BM and peritoneal macrophages, neutrophils, and mast cells, but it is not expressed on spleen lymphocytes. Data are representative of three experiments. The Journal of Immunology 5

Dickinson, Mountain View, CA), and data were analyzed with FlowJo Materials and Methods. Chimeric mice were bred to C57BL/6N software. mice to generate PILRa-deficient mice on a pure genetic back- Statistical analysis ground. Given the close proximity of PILRb, the neo-pgk cassette was removed by breeding PILRa-knockout mice to Rosa-26-cre Statistical significance was determined by the parametric Dunnett test for the knockin mice to avoid nonspecific effects on PILRb or other sur- comparison of joint cytokine concentrations, by the nonparametric Dunnett rounding (35). RT-PCR analysis revealed that PILRa mRNA test for the comparison of arthritis scores and histopathological as well as 2 2 micro-CT results, and by Student t test for the comparison of in vitro cytokine production was abolished in the spleen and BM of Pilra / mice, data. The p values , 0.05 were considered significant. whereas PILRb expression was not affected (Fig. 1C). Real time RT-PCR was done to better quantitate PILRb levels in WT and Results 2/2 2/2 Pilra mice. These expression studies showed that PILRb ex- Generation and characterization of Pilra mice pression in spleen and BM was not affected (Fig. 1D). PILRa To generate the PILRa-knockout mouse, exon 1 and exon 2 of protein was abundant in WT BM and was detected in spleen at the PILRa gene were deleted by homologous recombination in lower levels. However, in Pilra2/2 mice, no PILRa protein was C57BL/6J ES cells (Fig. 1A), and the targeted locus was con- detected in either BM or spleen, and Pilra+/2 heterozygous mice firmed by Southern blot analysis using internal (Fig. 1B, left displayed intermediate levels of protein (Fig. 1E). Pilra2/2 mice panel) or external (Fig. 1B, right panel) probes, as described in are viable, fertile, and have a normal lifespan. They did not show Downloaded from

FIGURE 3. Pilra2/2 mice are more susceptible to arthritis. (A) Immunohis- tochemistry of inflamed joint tissue (CAIA model, day 11). Within the cavity of an inflamed joint, PILRa is http://www.jimmunol.org/ expressed on neutrophils (arrows), the predominant cell type in CAIA. (B–E) C57BL/6N WT (n = 6) and Pilra2/2 mice (n = 8) were given arthritogenic anti–type II collagen mAb mixture to induce arthritis. (B) Pilra2/2 mice de- velop more severe CAIA. Pilra2/2 mice showed increased average clinical score (left panel), average daily clinical score (days 4–12, middle panel), and end by guest on September 24, 2021 point clinical score (day 12, right panel) compared with WT mice. (C)Hind paws, sagittal H&E. Representative images of the most severe lesions per group. Periarticular infiltration with in- flammatory cells and formation of intra- articular exudate are obvious in Pilra2/2 mice (arrows) compared with WT mice, where these features are essentially ab- sent. Scale bar, 200 mm. (D) Lesion scores for features of arthritis, including infiltration with inflammatory cells, fibroplasia, cartilage injury, and bone remodeling score per group on day 12, are lower in WT mice compared with Pilra2/2 mice. Data points represent average scores for two paws/mouse. Two limbs per animal, four to six sec- tions/animal, and a total of 14 mice were examined. (E) Hind paws from mice harvested on day 12 were subjected to micro-CT analysis. Images show high resolution three-dimensional rendering of micro-CT scans. Quantification of bone integrity, including JCBV and PNBV are shown. Results are represen- tative of two experiments. The micro- CT parameter estimates for Pilra2/2 mice were compared with the control group using the Dunnett test. 6 PILRa REGULATES INFLAMMATION any appreciable developmental defects in myeloid or lymphoid panel), littermate WT mice started to develop clinical signs of cell populations (Supplemental Table I). arthritis 5 d after injection of the type II collagen–specific Ab Previous work showed that PILRa transcripts are relatively high mixture, 2 d after LPS administration, and reached a maximum in spleen, liver, and lung (8). We tested PILRa protein expression clinical score of 3.8 6 2.6 (mean 6 SD) between days 7 and 9 in these tissues by immunohistochemistry (Fig. 2A) using Pilra2/2 before gradually resolving disease. Pilra2/2 mice showed similar mice as a negative control and in-house–generated Armenian kinetics of disease development as did WT mice, but they hamster anti-mPILRa mAb, which did not show cross-reactivity exhibited more severe disease, with maximum clinical scores of to mPILRb. In the spleen, PILRa+ cells are mainly localized in 8.0 6 2.6 (mean 6 SD). Pilra2/2 mice also had significantly the red pulp, consistent with expression predominantly by myeloid increased average clinical scores and end point clinical scores cells. In liver and lung, PILRa is expressed in tissue-resident compared with WT mice (Fig. 3B, middle and right panels). macrophages, such as Kupffer cells or exfoliated alveolar mac- On termination of the experiment at day 12, histopathology rophages, respectively. In BM, most PILRa+ cells are identified confirmed the clinical observations. Compared with WT mice, as granulocytes or their precursors. These data corroborate the Pilra2/2 mice had more severe disease, as shown by all evaluated notion that PILRa is expressed primarily in cells of the myeloid parameters, including number of affected joints/paw, periarticular lineage. Correspondingly, PILRa protein is detected on granulo- infiltration with mixed inflammatory cells, soft tissue proliferation cytes (CD11b+Ly6G+) and monocytes (CD11b+Ly6G2)fromBM, (including pannus formation), cartilage injury, and bone remod- as well as peritoneal cavity macrophages (CD11b+Gr-12), neu- eling (Fig. 3C, 3D). Consistent with the histological evidence for trophils (CD11b+Gr-1+), and mast cells (CD117+FcεRI+), but not more extensive bone remodeling, micro-CT revealed more severe on spleen lymphocytes, including T, B, NKT, and NK cells osteolysis (i.e., smaller JCBV), as well as increased new bone Downloaded from (Fig. 2B). formation (i.e., greater PNBV), in Pilra2/2 mice compared with 2 2 WT mice (Fig. 3E). These results support the regulatory role of Pilra / mice develop enhanced autoimmune arthritis PILRa in downregulating myeloid-dependent inflammation. To investigate the biological function of PILRa in myeloid cell– a mediated immune responses and its role in autoimmune diseases, PILR deficiency results in an increase in intra-articular proinflammatory cytokine levels at the onset of arthritis

we made use of a CAIA model, which is a myeloid-dependent http://www.jimmunol.org/ preclinical disease model. We observed an infiltration of PILRa- To better understand how PILRa deficiency impacts CAIA, we producing cells in mouse arthritic tissues, specifically periarticular tested inflammatory cytokine levels in joint tissue at the onset of soft tissue obtained from CAIA (Fig. 3A). We first tested how disease. Onset of disease was similar in WT and Pilra2/2 mice, CAIA is affected by PILRa deficiency. As shown in Fig. 3B (left with the start of clinical signs of arthritis on day 5 following in- by guest on September 24, 2021

FIGURE 4. Elevated levels of inflammatory cytokines were detected in joints of Pilra2/2 mice at the onset of disease. (A) C57BL/6N WT (n = 9) and Pilra2/2 mice (n = 9) were given arthritogenic anti–type II collagen mAb mixture to induce arthritis (CAIA). On day 5, hind footpads were obtained from mice for homogenization in cell lysis buffer containing protease inhibitors. Cytokine levels in lysate supernatants were analyzed by ELISA. The cytokine concentrations are expressed as pg/mg of protein. (B) Kinetics of neutrophils and macrophages in the early phase of CAIA show a similar degree of infiltration in arthritic joints of Pilra2/2 and WT mice. WT (dark gray bars, n = 4) and Pilra2/2 mice (light gray bars, n = 4) were given arthritogenic anti– type II collagen mAb mixture to induce arthritis. On days 3, 4, and 5, paws were collected for immunohistochemical staining of MPO and F4/80. Intensity of infiltration was estimated using an arbitrary scoring scheme ranging from 0 to 3. Bars represent mean of four arthritic paws. Error bars are SEM. Results are representative of two or three separate experiments. The p values are based on the parametric Dunnett test. The Journal of Immunology 7 jection of type II collagen–specific Abs. At this time point, hind paw lysates of Pilra2/2 mice exhibited significantly increased pro- duction of proinflammatory cytokines, including IL-1b and IL-6, as well as neutrophil and monocyte chemokines KC and MCP-1 (Fig. 4A). The increased inflammatory cytokine and chemokine production could be due to either increased inflammatory cell number or activity. It was shown that PILRa plays an important role during acute inflammatory responses in regulating neutro- philic migration (16). We then asked how cellular infiltrates are impacted in the ankle joints at early time points each day from day 3 to day 5 using immunohistochemistry MPO to stain neutrophils or F4/80 to localize macrophages (Fig. 4B). Neutrophils were slightly reduced in PILRa-knockout joints on day 3; however, their numbers were transiently increased on day 4 compared with WT mice. Macrophages in PILRa-knockout joints also showed a similar trend on day 3; however, their numbers were slightly increased on day 5. Overall, the changes in macrophage and neutrophil cellularities were not statistically significant between 2 2 WT and Pilra / mice (Fig. 4B). The slight reduction in neu- Downloaded from trophil/macrophages in PILRa-knockout mice on day 3 might be secondary to the systemic effect of LPS on the increased mi- gration of these cells to other tissues, such as liver, in these mice (16). In addition, we did not observe any difference between WT and Pilra2/2 neutrophil and macrophage cellular infiltrates in an

acute localized inflammatory model of Staphylococcus aureus FIGURE 5. Engaging PILRa on BMDCs reduces cytokine production. http://www.jimmunol.org/ A 2/2 (Supplemental Fig. 1). These observations support the notion that ( ) BMDCs express PILRa. WT and Pilra BMDCs were stained with B 2/2 the lack of PILRa has a global effect on the severity of arthritis, anti-mPILRa mAb or isotype control. ( ) WT and Pilra BMDCs were stimulated with mIgG plus various concentrations of mouse anti-mPILRa mainly through enhanced cytokine production. In summary, our 2/2 mAb–coated Protein G Agarose beads for 18 h, and cytokine production in data suggest that the joint-infiltrating Pilra myeloid cells are the supernatants was measured. Results are representative of three experi- functionally hyperresponsive, further highlighting the regulatory ments. *p , 0.05, Student t test. role of PILRa in myeloid cells. Engagement of PILRa suppresses the production of A recent study showed that PILRa associates with its ligands in

inflammatory cytokines and chemokines cis on neutrophils (16). Because BMDCs express both PILRa by guest on September 24, 2021 and its ligands (Supplemental Fig. 3A), we predicted that PILRa To confirm the regulatory role of PILRa in modulating cellular would interact with its ligands in cis, and this may play a role in function, we tested how the production of cytokines and chemo- negatively regulating BMDC function. If this were the case, dis- kines is affected in Pilra2/2 cells or upon its engagement in vitro. sociation of the interaction between PILRa and its ligands would We used various PILRa-expressing cell types, such as BMDCs, increase BMDC cytokine production. We showed previously that BM-derived monocytes (BMDMs), and neutrophils, which are sialic acid is an essential component of PILRa ligands in pri- implicated in the pathology of anti-CAIA. First, we tested how mary cells, and treatment of primary cells with sialidase A, which these cells respond to LPS, which is used to activate myeloid cleaves sialic acid from surface proteins, abolished their binding cells in the same CAIA model. The results showed that Pilra2/2 to PILRa (9). Therefore, we used sialidase A treatment to disso- BMDCs produced increased TNF-a and MCP-1 in response to ciate PILRa–ligand interactions on BMDCs. We found that sial- LPS but not IL-1b, IL-6, or KC (Supplemental Fig. 2). A subtle idase A treatment increased cytokine production by BMDCs in trend of cytokine elevation was seen in neutrophils and BMDMs response to mIgG coated on protein G beads, suggesting that the (Supplemental Fig. 2). The state of cellular hyperresponsiveness, interaction of sialylated ligands with their receptors inhibits cel- as measured by cytokines/chemokines, appears to vary in differ- lular function (Supplemental Fig. 3B). Given the fact that PILRa ent PILRa-expressing cells. BMDCs were more sensitive to this is able to interact with a network of ligands expressed by different pathway, which might be explained by the high expression of tissues, including hematopoietic cells other than myeloid cells (9), PILRa (Fig. 5A) in these cells. cis and trans interactions may both be involved in the regulation We then asked how engagement of PILRa receptor in BMDCs of PILRa-expressing myeloid cell function. modulated cytokine production. To couple stimulatory and puta- tive inhibitory pathways, we used mIgG-coated beads as the stim- a ulus. WT and Pilra2/2 BMDCs produced similar levels of the Anti-PILR mAb treatment attenuates arthritis proinflammatory cytokine TNF-a and chemokine MCP-1 in re- We then tested whether treatment with anti-PILRa mAb affects sponse to mIgG-coated beads (Supplemental Fig. 2). However, inflammation in vivo using the CAIA model. Because C57BL/6N stimulation of BMDCs with beads coated with anti-mPILRa mAb, WT mice develop only mild disease, we used BALB/c mice in- but not isotype control, showed a dose-dependent inhibition of TNF stead for anti-PILRa mAb treatment. We found that anti-PILRa and MCP-1 production in WT cells. The inhibitory effect of anti- mAb treatment significantly reduced inflammation compared with PILRa Ab was not seen in the BMDCs derived from Pilra2/2 cells, isotype control treatment (Fig. 6A). Isotype control–treated mice again confirming that the inhibitory effect is PILRa dependent (Fig. started to develop clinical signs of arthritis 4 d after injection of 5B). This result indicates that engagement of PILRa downregulates type II collagen–specific Ab mixture or 2 d after LPS admin- proinflammatory cytokine and chemokine production when it is istration and reached maximum clinical scores of 14.6 6 2.2 proximal to an activating receptor, such as FcgR. (mean 6 SD) between days 5 and 7. By contrast, anti-PILRa mAb– 8 PILRa REGULATES INFLAMMATION treated mice exhibited delayed disease onset, which gradually anti-PILRa mAb–treated mice exhibited a reduced maximum increased to a much reduced maximum clinical score of 4.4 6 4.3 clinical score of 8.5 6 4.0 (mean 6 SD) between days 5 and 7 (mean 6 SD) by the end of the study. Anti-PILRa mAb–treated (Fig. 6D, left panel). Anti-PILRa mAb–treated mice also showed mice also showed significantly reduced average clinical scores significantly reduced average clinical scores and terminal clinical and end point clinical scores compared with isotype control– scores compared with isotype control–treated mice (Fig. 6D, treated mice (Fig. 6A, middle and right panels). In accordance middle and right panels). Additional studies are required to un- with in vivo findings, histopathological assessment of hind paws at derstand how PILRa is relevant in the context of additional in- the termination of the study at day 11 showed significantly de- flammatory diseases. creased scores for all scored features in anti-PILRa mAb–treated a mice compared with isotype control Ab–treated mice (Fig. 6B). PILR is expressed in human inflammatory arthritic tissues Finally, micro-CT imaging of paws at day 11 showed that anti- and correlates with monocyte and DC levels PILRa mAb–treated mice exhibited less severe bone remodeling To test whether PILRa is involved in human inflammatory ar- compared with isotype control Ab–treated mice (Fig. 6C), as thritis, we assessed the expression patterns of PILRa and its coun- measured by JCBV and PNBV (p , 0.01 for both parameters). terpart PILRb using mRNA microarrays on human synovial tissue. The observation that anti-PILRa treatment attenuates arthritis Samples were obtained during joint resection surgery from OA and development, whereas Pilra2/2 mice show enhanced disease, sug- RA patients. Sections were taken for both histological observation gests that the anti-PILRa mAb does not block the receptor but and mRNA extraction. The Affymetrix expression microarrays in- rather engages it. To measure the impact of PILRa modulation on cluded two probes each for PILRa and PILRb.PILRa consistently other arthritis models, we tested the effect of anti-PILRa treat- showed significantly higher expression in the RA patients com- Downloaded from ment on mouse K/BxN serum–transfer arthritis. The K/BxN se- pared with the OA patients, including a correction for inflamma- rum–transfer model is a widely accepted model of inflammatory tion status (p , 1e27) (Fig. 7A, upper panels). In contrast, PILRb arthritis that shares features of human RA (25). In K/BxN mice, showed a modestly higher level of expression in OA patients (p , the induction of disease does not require LPS, and this disease 0.02) (Fig. 7A, lower panels). The differential expression of PILRa depends highly on the innate cells in a TNF-independent fashion and PILRb in inflammatory RA suggests that PILRa might func-

(36–39). Mice started to develop clinical signs of arthritis 1 d tion as a negative-feedback regulatory loop to dampen inflammation. http://www.jimmunol.org/ after K/BxN serum transfer. Isotype control–treated mice reached Cell-specific gene signatures can be used as an estimate of cellularity maximum clinical scores of 14.7 6 1.0 (mean 6 SD), whereas (40). For each cell type, we calculated a summary gene set score

FIGURE 6. Anti-PILRa mAb treatment attenu- A C

ates arthritis development. ( – ) BALB/c mice by guest on September 24, 2021 were given arthritogenic anti–type II collagen mAb mixture to induce arthritis (CAIA) and were treated with anti-PILRa mAb (n = 5). (A) Anti-PILRa mAb–treated mice exhibited reduced disease de- velopment compared with isotype control. Anti- PILRa–treated mice showed decreased average clinical score (left panel), average daily clinical score (days 4–11, middle panel), and end point clinical score (day 11, right panel) compared with isotype control–treated mice. (B) Pathological scores of isotype control and anti-PILRa mAb– treated mice on day 11. Data points represent av- erage scores for two paws/mouse. Two limbs per animal, four to six sections/animal, and a total of 10 mice were examined. (C) Hind paws from mice harvested on day 11 were subjected to micro-CT analysis. Images show high-resolution three-di- mensional rendering of micro-CT scans. Quantifi- cation of bone integrity, including JCBVand PNBV. (D) Anti-PILRa mAb treatment inhibited disease development in a K/BxN serum–transfer arthritis model. BALB/c mice were given 20 ml of arthro- genic K/BxN serum on days 0 and 2 by i.v. injec- tion and were treated with anti-PILRa mAb (n = 6). Mean clinical score (left panel), average daily clinical score (middle panel), and day-7 clinical score (right panel) are shown. In vivo arthritis scores and micro-CT parameter estimates for the treatment groups were compared with the control group using the Dunnett test. Results are repre- sentative of two experiments. The Journal of Immunology 9 using a quartile-trimmed mean of the normalized probe set values not significantly correlated with neutrophil-specific genes (r = 20.1, present in the gene set. This summary score provides an estimate for p , 0.3) (Fig. 7B, lower panels). These trends held true when we the relative abundance of the given cell type across samples. Cor- considered all samples together or RA and OA samples in isolation. respondingly, we found that PILRa expression was highly correlated Our human expression study shows that PILRa expression may with the tissue-specific gene sets obtained from different inflam- dissociate from PILRb, and this inhibitory pathway is overrepre- matory cell types, such as DCs (Spearman r =+0.7,p , 2e216), sented in myeloid cells in human RA. neutrophils (r =+0.5,p , 2e25), and monocytes (r =0.8,p , 2e2 a 16) in RA samples (Fig. 7B, upper panels). In contrast, PILRb Diverse ligands are involved in PILR pathway expression was minimally inversely correlated with DC-specific and We (9) and other investigators (8, 10, 41) reported that PILRa monocyte-specific genes (r = 20.2 for both, p , 0.01), and it was engaged multiple ligands with common molecular determinants Downloaded from http://www.jimmunol.org/ by guest on September 24, 2021

FIGURE 7. PILRa-inhibitory versus PILRb-activating pathways in human RA joints and their correlation with pathogenic cell types. Synovial samples were taken from RA (n = 81) and OA (n = 121) patients during joint resection surgery. (A) The Affymetrix expression microarrays included two probes each for PILRa and PILRb. PILRa expression is significantly higher (p , 1e207) in RA patients compared with OA patients; this also holds when corrected for inflammation status. PILRb expression is modestly higher in OA patients compared with RA patients (p , 0.02), correcting for inflammation status. (B) The Affymetrix expression microarrays of PILRa and PILRb. In RA patients, PILRa expression is significantly correlated with DC-specific genes (r = +0.7, p , 2e216), neutrophil-specific genes (r = +0.5, p , 2e25), and monocytes (r = +0.8, p , 2e216). This trend also holds true for all of the in- flammatory RA subset of patients. In contrast, PILRb expression was not positively correlated with DC, neutrophil, or monocyte gene expression (r = 20.2, p = 0.01; r = 20.1, p = 0.2; and r = 20.2, p , 0.01). Scores were calculated using a quartile-trimmed mean of the normalized probe set values present in the gene set. 10 PILRa REGULATES INFLAMMATION

(9). Using recombinant hPILRa-Fc to screen a high-throughput with individual ligands, it is interesting that a conserved domain human full-length cDNA expression library, we identified an ad- in PILRa is critical for its interaction with all known ligands, ditional PILRa ligand CLEC4G [previously named LSECtin (42)] a property shared with the Siglec family of inhibitory receptors (Fig. 8A). We confirmed its binding to both human and mouse (9, 43). A recent study showed that PILRa interacting domain in PILRa by transfecting HEK 293T cells with this putative ligand cis engaged its ligands in the same cell (16). The new ligand and staining with human and mouse PILRa-Fc for analysis by CLEC4G is expressed in macrophages, it might be able to interact flow cytometry (Fig. 8B). Consistent with our previous find- in cis with PILRa. CLEC4G is implicated in Ebola virus entry ings that an evolutionarily conserved PILRa arginine site receptor, as well as clearance and presentation of various Ags (hPILRaR126A and mPILRaR133A) is essential for its interac- (44). Given the complexity of the PILRa receptor/ligand net- tion with diverse sialylated ligands, we found that hPILRaR126A- work, PILRa appears to have a diverse function in that it fine Fc and mPILRaR133A-Fc lost their binding activity to these tunes the immune response depending on inflammatory cues, ligands (Fig. 8B), suggesting that they all share the same sialy- similar to other paired receptors (45). Additional studies are re- lated PILRa binding domain. mPILRb-Fc also bound to CLEC4G quired to determine how cis/trans interactions of PILRa ligands (Fig. 8B, far right panel). Thus, a complex network of ligands are modulated during inflammation where this pathway is up- using a common molecular determinant may modulate the PILRa regulated. pathway in different tissues. In the context of inflammatory arthritis, the main effect of PILRa knockout is identified as an enhanced production of pro- Discussion inflammatory cytokines, resulting in more severe disease. At the In this study, we reported that PILRa represents a potent inhibitory onset of arthritis, PILRa-knockout mice produced higher levels Downloaded from receptor that negatively regulates the function of myeloid cells of proinflammatory cytokines, such as IL-6 and IL-1b, as well as to control inflammation in mouse arthritis models. PILRa is chemokines, such as KC and MCP-1, in joint tissues, even though 2 2 predominantly expressed by macrophages, DCs, and neutrophils, both WT and Pilra / cellular infiltrates in arthritic joints were 2 2 all of which are important in the pathogenesis of arthritis. Using similar in WT and Pilra / mice by histopathology. This suggests PILRa-knockout mice in a CAIA model, we demonstrate that the that, in the absence of PILRa, myeloid cells infiltrating the absence of PILRa leads to dysregulation of inflammatory pro- joints are hyperresponsive, supporting a regulatory role for PILRa http://www.jimmunol.org/ cesses in inflamed tissues, resulting in increased inflammatory in these cells. Although the myeloid cells in joint tissues at the cytokine production and severity of disease. Conversely, engaging onset of disease are hyperresponsive in terms of proinflamma- PILRa with an anti-PILRa mAb reduces disease severity in both tory cytokine production, we did not detect robust hyperrespon- CAIA and K/BxN serum–transfer models. siveness of PILRa-expressing cells in the absence of PILRa 2 2 In preclinical arthritis models, as well as in human inflammatory in vitro. Pilra / BMDCs produced mildly, but consistently, in- arthritis, PILRa gene expression profiling supports the notion that creased levels of TNF and MCP-1 in response to LPS, whereas WT 2 2 this receptor acts in a counter-regulatory fashion to dampen in- and Pilra / BMDCs produced similar levels of these cytokines in flammation. It is interesting that PILRa (inhibitory receptor), but response to mIgG binding to Protein G beads (Supplemental Fig. 2).

2 2 by guest on September 24, 2021 not PILRb (activating receptor), expression is positively corre- For BMDMs, Pilra / mice showed a trend toward increased lated with monocytes or neutrophil gene signatures in human RA, proinflammatory cytokine and chemokine production in response to suggesting that this pathway is highly regulated via its ligands to LPS (Supplemental Fig. 2). Therefore, the cumulative effect of this possibly keep these inflammatory cells in check. The usefulness augmented response may explain the robust phenotype in the context of the PILRa pathway in human RA or additional inflammatory of experimental arthritis. The inhibitory effect of PILRa receptor models requires addition studies. mediated by Ab cross-linking in vitro suggests that, when proximal, A complex network of ligands expressed in a variety of tissues stimulatory pathways, such as FcgR, can be diminished. PILRb was has been implicated in PILRa interactions (8–10). Although we do shown to promote cytokine production in myeloid cells, and 2 2 not fully understand the consequence of the interaction of PILRa PILRb / mice and BMDMs produced fewer proinflammatory cytokines (14). In addition, PILRa is able to regulate neutrophil infiltration during acute inflammation via modulation of integrin activation in an acute inflammatory peritonitis model (16), in which PILRa-knockout mice show increased infiltration of neu- trophils upon thioglycollate challenge (16). In contrast, in CAIA we did not see a difference between WT and PILRa-knockout mice with regard to the number of neutrophils and macrophages in ar- thritic joints. In support of this finding, we did not observe any difference between WT and Pilra2/2 neutrophil and macrophage cellular infiltrates in an acute localized inflammatory model of S. aureus (Supplemental Fig. 1). Despite these differences, both studies suggest that PILRa plays a negative regulatory role in the inflammatory process, likely by complex mechanisms under dif- FIGURE 8. CLEC4G is a novel ligand of PILRa.(A) Recombinant ferent inflammatory cues. Our findings suggest that, in the context hPILRa-Fc was screened for interacting partners using a Genentech hu- of sterile inflammation, PILRa is a regulatory receptor that mod- man full-length cDNA expression library. COS-7 cells were transfected ulates inflammatory cytokine responses; however, we cannot rule with individual cDNA expression vector and then exposed to PILRa-Fc, out that PILRa might have additional functions outside of cytokine followed by fixation. Protein–protein interactions were detected with an Alexa Fluor 488–conjugated anti-mouse IgG2a secondary Ab and scanned production to affect inflammatory responses. with an MD Isocyte plate scanner. (B) HEK 293T cells were transfected Anti-PILRa treatment reduced inflammatory arthritis. Consis- with expression vectors for CLEC4G and then stained with h/mPILRa-Fc, tent with this observation, engaging PILRa on BMDCs, which hPILRaR126A/mPILRa-R133A-Fc, or mPILRb-Fc (black line) or isotype highly express PILRa on their surface, inhibits their function control IgG (gray line). when a stimulating receptor, such as FcgR, is coengaged. Thus, The Journal of Immunology 11

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