Posted on Authorea 15 Apr 2020 — CC BY 4.0 — https://doi.org/10.22541/au.158696001.15833660 — This a preprint and has not been peer reviewed. Data may be preliminary. oee,rcn tde aesonta h niec n rvlnertsi oeAincutisare countries Asian some in rates prevalence and remission incidence of the periods that and shown increasing (CD), inflammation rapidly have disease by studies characterized Crohn’s recent disorders and However, chronic (UC) intestinal colitis relapse of ulcerative and group encompasses which a (IBD), comprises disease cells. bowel U937 in Inflammatory modulator anti-inflammatory an as functions and Introduction IBD We to pathways. related signaling is MEK/Erk LILRA3 and expression. that CD36 Akt time increasing of first by combination the phagocytosis a for secretion monocyte through report cells enhanced in proliferation and U937 monocyte decreases chemokines promoted significant of several LILRA3 to of behaviors Furthermore, led expression biological the monocytes the reducing in by on LILRA3 gration line LILRA3 of found, cell overexpression of was monocyte that effects interferon- development U937 found of the IBD Human We and with controls. explored. cells polymorphism healthy systematically with LILRA3-overexpressing the were compared of establish expression patients association to LILRA3 IBD no employed the in Despite was detect increased markedly to samples. was employed intestinal quantitative expression study, were and LILRA3 our (IHC) blood in immunohistochemistry controls patient healthy and IBD 509 be- blot in and and biological patients western polymorphism on IBD (qRT-PCR), LILRA3 effects 378 between PCR its in association whereas real-time assessed the monocytes, investigate was To in polymorphism expressed LILRA3 reported. susceptibility, inflammatory mainly systematically IBD of is been pathogenesis not LILRA3 have on yet. effects monocytes clarified its of However, been haviors immunoglobulin not leukocyte disorders. have autoimmune molecule (IBD) many anti-inflammatory disease with novel bowel associated the is of (LILRA3) deletion A3 6.7-kb receptor homozygous like a that shows evidence Growing Abstract 2020 28, April 4 3 2 1 Zhao Qiu and h ekct eetrcutro ua hoooe1.Tefml nlds1 ebr ihactivating with members 13 includes ) family The ( receptors re- 19. hibitory like chromosome immunoglobulin capacity human inhibiting Leukocyte on with or cluster IBD, disorders. receptor of autoimmune leukocyte pathogenesis multiple the the in in genes roles ( these important ceptors of play factors some genetic of that involvement indicated have studies Numerous Lan Xiucai an as functions and patients modulator anti-inflammatory IBD in increased is LILRA3 aiy eoi eunigo IR3hsrvae htLLA shgl oooost te IR such LILRs other to homologous highly is LILRA3 that revealed has LILRA3 of sequencing Genomic family. [6,7] hnhiIsiueo aei eiaCAS Medica Materia of Institute Shanghai Hospital Zhongnan University Wuhan Hospital Central Xuhui Shanghai University Fudan Hospital Zhongshan . LILRA3 LILRs γ [1] (IFN- 1 h ouain fwsencutisaemr ieyt ue rmIDta hs nAsia. in those than IBD from suffer to likely more are countries western of populations The . egu Liu Fenghua , IT6 D5) oae ntecnrmrcITcutr saseilmme fteLILR the of member special a is cluster, ILT centromeric the in located CD85e), (ILT-6, 3 yoys L,LR D5 r h otcnevdgnsaogtoelctdwithin located those among genes conserved most the are CD85) LIR, ILT, synonyms: ; γ [2-4] ,tmrncoi factor- necrosis tumor ), LILRBs . [5] IR ihln yolsi al ern yoiebsdihbtr oisaein- are motifs inhibitory tyrosine-based bearing tails cytoplasmic long with LILRs : ,weesLLswt hr yolsi al aeatvtn ucin ( functions activating have tails cytoplasmic short with LILRs whereas ), 2 igChang Ying , α (TNF- α 3 n nelui I-) diinly IR3aae ooyemi- monocyte abated LILRA3 Additionally, (IL-6). 6 interleukin and ) igLiu Jing , 1 3 ihaLan Xiuhua , 4 iXiang Li , 3 egZhou Feng , LILRAs 3 , Posted on Authorea 15 Apr 2020 — CC BY 4.0 — https://doi.org/10.22541/au.158696001.15833660 — This a preprint and has not been peer reviewed. Data may be preliminary. eecletdfo 4hatyvlner;aohr2 omlbose Zoga optlfo September from Hospital (Zhongnan samples biopsies mucosa normal colonic 20 Non-inflamed another All volunteers; ulcerations. Hospital. healthy to Zhongnan 14 tissues at adjacent colonic endoscopy from the sites blood underwent and/or collected who inflammatory for from ileal were patients from extracted Inflamed UC data taken 52 was Germany). sub-phenotype were and DNA Shanghai, patients biopsies genomic and (Qiagen, CD Total 36 kit demographic The from midi 1. collected selected The blood Table were DNA were Supplementary 2016. QIAamp in controls 2015. January the shown Healthy using November to are Gastroenterology, controls to 2014 criteria. of and January classification September Department patients from Montreal the from CD, volunteers from to China) (185 recruited according among (Wuhan, patients were sub-genotyped University IBD patients were Wuhan 378 All patients of from controls. obtained Hospital healthy were Zhongnan 509 samples and blood UC) peripheral 193 anti-coagulated sulfate Lithium Helsinki, Informed of collection (2014037). Declaration University samples the Wuhan and of in Patients Hospital expressed Zhongnan principles obtained. of the been committee with has ethics consent the accordance by in approved conducted was and been has study Our as Statement well Ethics as monocytes LILRA3- of establish Methods functions proliferation. to and above and the line Materials apoptosis on cell as LILRA3 monocyte such blood of human behaviors the effects biological U937 crossing the required other explored system, the are vascular then employed injury and We the cells of outside overexpressing reported. functions site systematically their the of been phagocytizing to many not directly migrating exert by and also surveillance wall cells immune vessel these in As roles IL-10. (DCs) and important cells mono-myeloid have circulation in dendritic and in expressed and pathogens responses mainly (Mø) immune is macrophages in LILRA3 monocytes, gulators interac- found, patients. was the as IBD investigated association in such we significant LILRA3 study, no cells, this increased Although in observed development. Accordingly, surprisingly IBD pathogenesis. we and IBD polymorphisms hypothesized in LILRA3 we role between inflammation, pathwaytion a intestinal to recurrent Erk/MEK play reported by might the characterized also through LILRA3 disorder formation that is autoimmune synapse an LILRA3 promote is inflammation, to IBD Because and from LILRB2 Apart of antagonist LILRA3. an of as function effect anti-inflammatory an been suggesting CD8 has of it factor- proliferation Additionally, induces necrosis diseases diseases. LILRA3 tumor inflammatory autoimmune whereas chronic for ( of monocytes, gene 10 pathogenesis human susceptibility the interleukin novel in that role a reported crucial is a LILRA3 play homozygous that might the and indicate with findings those These than expression, (SLE) inflammation. levels protein higher erythematosus and much lupus mRNA having mic (+/+) LILRA3 type affects (-/-) wild deletion deletion the 6.7-kb carrying the individuals case- that prostatic with demonstrated a benign have In with articles associated gene. recent be LILRA3 to the reported cancer state of was rs103294 (SNPs) population, polymorphisms Chinese hyperplasia single-nucleotide the among two study are control rs410852 and conserved rs103294 are which Sj LILRs, as other such diseases, to exons seven opposed first the as passing variation, presence-absence shows genetically LILRA3 addition, In LILRB2 and LILRB1 as [9] [15] [16] o xml,sm niiul a ar naern eeino .-bfamn encom- fragment 6.7-kb a of deletion aberrant an carry may individuals some example, For . [17-19] eoewd soito td GA)as dnie s024a e iklcsfrpro- for locus risk new a as rs103294 identified also (GWAS) study association genome-wide A . hsGA lorvae htplmrhs fti ou ffcsLLA xrsin Many expression. LILRA3 affects locus this of polymorphism that revealed also GWAS This . oehls,icesdLLA sdtce nmn iess uha Sadsyste- and MS as such diseases, many in detected is LILRA3 increased Nonetheless, . ge’ ydoe(S,mlil ceoi M)adremti rhii (RA) arthritis rheumatoid and (MS) sclerosis multiple (SS), syndrome ¨ ogren’s [26] [8] ugsigta IR3mgtatb marn h ucino hs LILRBs. these of function the impairing by act might LILRA3 that suggesting , ooye ert ayctknssc sIFN- as such cytokines many secrete Monocytes . [8,10] IL-10 n hsvrainhsbe rvnt eascae ihmn autoimmune many with associated be to proven been has variation this and , [18,19] + rinterferon- or ) oho hc r uomuedsrescaatrzdb excessive by characterized disorders autoimmune are which of both , -el n Kclsi h rsneo r-namtr cytokines pro-inflammatory of presence the in cells NK and T-cells α ( γ 2 ΤΝΦ-α ( ΙΦΝ-γ [27] xiisteopst effect opposite the exhibits ) h ffcso IR3o ooye have monocytes on LILRA3 of effects The . hrl peuae IR3epeso in expression LILRA3 upregulates sharply ) [21,23-25] γ, TNF- ooye r rtclre- critical are Monocytes . α, nelui ( 6 interleukin [20] Furthermore, . [11-14] IL-6 . [22] [21] ), . , Posted on Authorea 15 Apr 2020 — CC BY 4.0 — https://doi.org/10.22541/au.158696001.15833660 — This a preprint and has not been peer reviewed. Data may be preliminary. 2tasetdU3 el sn itr fRP yi ouincnann rtaeihbtradphos- and inhibitor protease containing solution lysis RIPA of mixture a using pWSLV- or cells plasmid- pWSLV-02-LILRA3 and U937 biopsies intestinal 02-transfected fresh-frozen from extracted was protein Total blotting Western (GAPDH). dehydrogenase SYBR glyceraldehyde-3-phosphate the gene with housekeeping 2 USA) The Carlsbad, Japan). Scientific, Kusatsu, Flex (ABI, (Thermo QuantStudioTM6 instrument the spectrophotometer using PCR performed NanoDrop2000 subsequently Real-Time was a qRT-PCR and USA). using Waltham, quantity Scientific, (1 assessed (Thermo The RNA were USA). Total samples or Carlsbad, USA). plasmid- RNA (Invitrogen, Waltham, pWSLV-02-LILRA3 reagent the and Trizol of biopsies the fresh-frozen quality using blood, cells peripheral (qRT-PCR) U937 was from reaction pWSLV-02-transfected extracted FCM chain was and polymerase RNA hours, time Total 72 real integration. quantitative for lentivirus and lentivirus LILRA3 extraction prepare with stable To RNA with infected transfection. cells after were hours select cells Kit 48 to Packaging U937 and used Expression hours then line, HIV of 24 Lenti-Pac cell at sequence using LILRA3-overexpressing collected intact control cloned was the a the Virus as and production, encodes USA). pWSLV-02 qRT-PCR which lentivirus reporter Rockville, vector empty For plasmid, (GeneCopoeia, a using pWSLV-02-LILRA3 the pWSLV-02-LILRA3. as or cells the plasmid (EGFP) cDNA, with U937 the LILRA3 protein co-transfected generate from fluorescent were to cells amplified green China) 293T enhanced was Beijing, (carrying LILRA3 (Viewsolid, pWSLV-02 of vector gene) expression (ORF) the frame into reading open line cell The (100 U937 medium penicillin LILRA3-overexpressing USA), DMEM the were Logan, in of cells (HyClone, cultured Establishment U937 (FBS) were The serum cells (100 China. bovine streptomycin Wuhan, 293T 10%fetal and in the were USA); U/ml) Collection containing of FDCC-HSN190) Logan, Culture USA) Identification Type (HyClone, Logan, (Cat. for China). medium (HyClone, Centre cells (Shanghai, RPMI-1640 China 293T University with the Fudan and at cultured of FDCC-HLC063) were (F.D.C.C) lines Center cell (Cat. two Cell line Fudan cell from monocyte obtained ABI U937 the human using assessed The were products LDR the culture and (PCR- Cell reaction, LDR reaction USA). the Carlsbad, detection in (ABI, template (Shanghai, PCR-ligation sequencer the Company 3730 the as Biotechnology used Applied by was Biowing detected product Shanghai were from support rs410852 China) technical and with method rs103294 reactions. LDR) separate of in in genotypes primers shown The are of electrophoresis sets gel genotyping two of amplified rs410852 results the region) and The using 6.7-kb rs103294 gel. tested the agarose was within 1.5% sample (designed a 1. on 2 Figure DNA analyzed Supplementary primer Each amplified were present, deletion) products was bp. the amplified LILRA3 (flanking 250 The 1 When of of primer sets fragment bp. deletion, two a 166 LILRA3 the With using of (PCR) fragment 2. reaction Table a chain Supplementary in polymerase by listed detected primers was was UC variation presence-absence and LILRA3 CD of with Diagnosis endoscopy genotyping and deletion China. examinations 6.7-kb radiological Central and from laboratory population symptoms, Han clinical controls. of histology. as the served combination of neoplasms a or were on polyps based controls from centimeters and 20 sites patients uninfected from All obtained 2015) June to 2014 [28,29] h eune ftepiesadpoe r itdi upeetr al .TePCR The 2. Table Supplementary in listed are probes and primers the of sequences The . -CT ehdwsapidt eemn h eaiemN eesnraie othe to normalized levels mRNA relative the determine to applied was method μ /l nhmdfid5 CO 5% humidified in g/ml) μ )wsue osnhsz DAuigafis-tadcN ytei kit synthesis cDNA first-strand a using cDNA synthesize to used was g) 3 2 t37 at ° C. ® rmxE aT Imx(Takara, mix II TaqTM Ex Premix Posted on Authorea 15 Apr 2020 — CC BY 4.0 — https://doi.org/10.22541/au.158696001.15833660 — This a preprint and has not been peer reviewed. Data may be preliminary. et.TeQ ra(nei -Epstv n -A eaie ersnseryaottccls n Q2 and cell cells, of apoptotic sign early apoptosis. a represents late fragments, negative) represents DNA with 7-AAD positive) label 7-AAD phosphatidylserine and twice to and positive detect San washed used V-PE V-PE to (BD, was and annexin (annexin 7-AAD used kit (both harvested area and was (7-AAD) were Q4 apoptosis, V-PE D The cells early V-PE/7-amino-actinomycin Annexin death. of the annexin protocols. hallmark culture, an manufacturer’s a of using the externalization, following stained hours then USA) 48 were Jose, After cells plates. The 6-well cells PBS. U937 in cells. U937 plated of apoptosis were in LILRA3 of involvement the detect integrated(4 to applied the was (FCM) calculate cytometry Flow to #IX73). used Cat. was (Olympus, assay MD) microscope Apoptosis a BetheSEMa, primary by the Cybernetics, acquired of (IOD). (Media were instead density Images 6.0 option antibody (PBS) control. version primary saline negative 5% Plus the phosphate-buffered a with with Image-Pro with as incubated treated used blocking were were Sections by sections antibody. antibody followed The secondary buffer, a temperature. citrate with room antigen sodium at 4 and minutes deparaffinized, at using were overnight 30 irradiation sections for The oven serum microwave individuals. for goat control 3- by protocols and embedding, standard retrieved IBD with paraffin with was accordance patients After in from 3 (H&E) on tissues formalin. eosin performed colon and was 10% hematoxylin IHC in with examination. overnight stained microscopic and fixed cut were were histopathology sections for biopsies Colon immunofluorescence according and China), (IHC) Shenzhen, immunohistochemistry (NeoBiosciences, Histopathology, kits ELISA protocol. manufacturers’ using the measured to was 20,000 CXCL10 at and centrifuged enhanced -80 CXCL8/IL-8 and at using collected stored were visualized and cells aliquoted were U937 blots of media the Culture TBST, with washes 3 Elisa After USA). temperature. Hercules, TBST (HRP)-conjugated (Bio-rad, room in the peroxidase kit China) at as horseradish chemiluminescence Tianjin, hours used Biotechnology, a Sungene was 2 with LK2001/LK2003, China) incubated for (Cat. Tianjin, then antibody Technology Biotechnology, were anti-rabbit/mouse Signaling Sungene membranes goat Cell #3033), KM9002, The from purchased (Cat. (Cat. reference. were anti-p- GAPDH internal mAb #2983) #9258), USA). (Cat. anti-p-P65 (Cat. Danvers, mAb mAb #4257), anti-mTOR anti-JNK2 (CST, #5536), #4668), (Cat. (Cat. (Cat. mAb mAb mAb (Cat. anti-p-SAPK/JNK mTOR anti-PI3K pAb #8242), #4228), anti-p-c-Raf (Cat. #3062), mAb (Cat. (Cat. anti-P65 MAPK pAb pAb anti-p-Erk1/2 anti-P38 anti-PDK1 #4511), #3438), anti-p-PI3K #8727), (Cat. (Cat. (Cat. #9421), mAb mAb MAPK anti-p-PDK1 mAb anti-p-P38 #8690), anti-Akt anti-MEK1/2 #4695), (Cat. (Cat. #4060), #9154), mAb mAb (Cat. (Cat. anti-Erk1/2 (mAb) #4370), mAb (Cat. antibody anti-p-MEK1/2 mAb monoclonal #4691), were anti-p-Akt (Cat. ab17026) USA); (Cat. mAb (Cambridge, mAb anti-Foxo3a from Abcam and obtained from were ab47285) 18704-1-AP) procured (Cat. Pkwy (Cat. mAb antibodies pAb anti-p-Foxo3a Alton anti-CD206 primary GeneTex, China); and (Wuhan, GTX108819, specific 18836-1-AP) Proteintech (Cat. (Cat. with (pAb) pAb incubated anti-CD36 antibody then USA); polyclonal Irvine, and anti-LILRA3 (TBS) hour antibodies: saline 1 following (Tris-buffered 4 for the TBST at temperature in overnight room milk TBST nonfat at in (Millipore, 5% 20) membranes with Tween (PVDF) un- blocked 0.1% (SDS-PAGE) difluoride were with electrophoresis polyvinylidene membranes onto gel The transferred sulfate-polyacrylamide USA). then dodecyl Burlington, kit and sodium BCA 40 conditions 8% the of reducing and or total der RIPA 12.5% A kit. by China). assay separated BCA (Shanghai, was a Biotechnology using Beyotime determined from was purchased concentration were protein The inhibitor. phatase × 10 5 el/el abrn h ulvco pSV0)adoeepeso lsi (pWSLV-02-LILRA3) plasmid overexpression and (pWSLV-02) vector null the harboring cells/well) ° .Atrcrflwsig h etoswr hnicbtdfr1hu tro temperature room at hour 1 for incubated then were sections the washing, careful After C. ° olwdb he ahswt BT h ebae eete nuae with incubated then were membranes The TBST. with washes three by followed C ° ni nlss h ocnrto fINgma L6 TNF- IL-6, IFN-gamma, of concentration The analysis. until C μ etoso aanebde rs-rznendoscopic fresh-frozen paraffin-embedded of sections m 4 × o i.Tespraat were supernatants The min. 1 for g μ rti rmec sample each from protein g α, C2 CCL3, CCL2, μ m Posted on Authorea 15 Apr 2020 — CC BY 4.0 — https://doi.org/10.22541/au.158696001.15833660 — This a preprint and has not been peer reviewed. Data may be preliminary. p09 o s024 =.7frr405) oehls,a h leelvl h leeTo s024was rs103294 of T allele the level, allele the susceptibility. disease at and Nonetheless, genotypes rs410852). rs410852 for and p=0.17 rs103294 rs103294; the China. for between Central association (p=0.94 of detected no has population was deletion Han association 6.7-kb the the Limited among that indicate development data UC These and 2). (Table CD UC with or CD of frequencies phenotype p (p=0.27, p development (“-” p=0.87, level IBD CD; allele and the deletion at p 6.7-kb difference p=0.39, no the CD; (89.4% between (CHB) for found Beijing 95%CI=0.78-2.47 was 89.4%, in OR=1.39, was association population controls) no Chinese healthy Han and and the UC, patients for (both reported study that with our vs. comparison in IBD in enrolled higher of subjects was development 877 which among the rate and deletion polymorphism significant. The gene statistically LILRA3 be between to association considered The was 0.05 were than plasmid less and overexpression value Results mRNA and p mean LILRA3 vector A as null in presented t-test. are the Differences variance unpaired Data of confidence harboring UC. analysis an 95% cells one-way and using using with sub- U937 CD analyzed provided analyzed the the were developing are controls between between of and (ORs) differences Differences groups risk ratios (ANOVA). patient the relative Odds the and between the expression controls. controls estimate protein the and to with Hardy- cases (CI) groups in the interval patient were between the rs410852 genotypes of and and phenotypes The rs103294 alleles respectively). the genotype, Among p=0.55 of deletion 5.0. p=0.31, frequencies 6.7-kb version (p=0.55, software the (HWE) Prism of GraphPad Equilibrium Weinberg and distributions 17.0 the Version controls, SPSS using healthy performed were analyses were All replicates parallel Three analysis Switzerland). Statistical Manedorf, Tecan, 37 sample. at M200, each incubation in for (Infinite After employed plated reader time. were indicated auto-microplate vector the an CO at manufacturer’s null 5% added the the were humidified to Japan) or in Kumamoto, according plasmid gun assay overexpression kamimashiki (CCK-8) (Dojindo, LILRA3 kit-8 (3.0 the counting plates harboring cell 96-well cells the U937 by detected instructions. was proliferation Cell assay proliferation Cell GFP of percentage hours. the The 24 well. on each LILRA3 to of added influence seeded were potential pWSLV-02 the or pWSLV-02-LILRA3 37 (1.0 detect harboring to plates lines cell used 6-well U937 were in cells. USA) U937 Louis, of capacity St. phagocytosis Sigma, (L3030, 37 beads at Latex hours 6 chamber. for counting assay cell incubation Phagocytosis a After using counted were chamber. cells, lower migrated the to CO added 5% with was USA) medium Corning, complete (Corning, chambers Boyden 24-well 5 transwell using 8.0 performed an was assay migration A migration Cell × ° 10 nhmdfid5 CO 5% humidified in C 70%) 4 el/eli 200 in cells/well μ 2 oesz ebaewtotmtie.Clswr eddi h pe hme tadniyof density a at chamber upper the in seeded were Cells matrigel. without membrane size pore m h pe hme a eahdfo h el n h el ttelwrcabr representing chamber, lower the at cells the and well, the from detached was chamber upper the , [30] h eeinrtoi he rusws8.%frhatycnrl,9.%frC n 07 for 90.7% and CD for 91.4% controls, healthy for 88.4% was groups three in ratio deletion The . FDR 08,O=.2 5C=.013 o C Tbe1,adtedlto i o ffc the affect not did deletion the and 1), (Table UC) for 95%CI=0.80-1.30 OR=1.02, =0.87, × × 10 2 10 3 μ o or h pia est O)o ahsml a esrda 5 musing nm 450 at measured was sample each of (OD) density optical the hour, 1 for el/el o ,1,2,4 n 2hus oec ape 10 sample, each To hours. 72 and 48 24, 12, 0, for cells/well) eu-reRM-60mdu.A hmatatn,600 chemoattractant, a As medium. RPMI-1640 serum-free l 5 el/el nRM-60mdu otiig1 B,floe yicbto at incubation by followed FBS, 1% containing medium RPMI-1640 in cells/well) 2 o 0hus itr fFSadltxbas(B:ltxbas=2:)was 20:1) = beads latex (FBS: beads latex and FBS of mixture A hours. 20 for ± Dfo he needn xeiet n ersnaiedt r shown. are data representative and experiments independent three from SD vs. +)wsfud(=.0 p (p=0.70, found was “+”) + ea Red Texas FDR 09,O=.8 5C=.322 o C.I addition, In UC). for 95%CI=0.73-2.22 OR=1.28, =0.96, 5 + el a hnaaye yFMa .,6 2and 12 6, 0.5, at FCM by analyzed then was cells FDR 09,O=.5 5C=.213 for 95%CI=0.82-1.35 OR=1.05, =0.90, χ etwsue oaayethe analyze to used was test 2 μ 0 B containing FBS 10% l μ C- solutions CCK-8 l ° nhumidified in C FDR =0.41, ° C Posted on Authorea 15 Apr 2020 — CC BY 4.0 — https://doi.org/10.22541/au.158696001.15833660 — This a preprint and has not been peer reviewed. Data may be preliminary. IR3pouto nvto u eut upr h rwn oyo vdneta IR3participates LILRA3 that evidence of demonstrated body have TNF- growing but the studies upregulates support Previous IL-10 results 3C). that Our (Figure findings vitro. inhibition on in results, based of previous production LILRA3 effect the U937 LILRA3 IFN- for with significant LILRA3-overexpressing consistent role of use more in anti-inflammatory and secretion we a an hours, protein, the showed 24 soluble and in hours 12 a decreased 12 for is the cells significantly of LILRA3 U937 inflammation, detected presence the the the in we culture that to that role of supernate demonstrated view lines its assay explore cell In elucidate IFN- To Elisa 3B). of further collected. cells. (Figure secretion were U937 and 0.001) downregulate lines overexpressing secretion markedly cell the could cytokine established in LILRA3 two on detected the LILRA3 shown were of As of LILRA3 supernatant levels. effect protein in and potential increases mRNA significant the the 3A, at cells verified Figure U937 was development. in by cells IBD U937 cytokines in LILRA3-overexpressing many LILRA3 of of Establishment of secretion role macrophages downregulates definite in a LILRA3 expressed identifying of is patients, Overexpression protein IBD CD68 LILRA3 in more of increased and majority remarkably possessed overwhelming is CD68 patients the and use CD that demonstrate then and LILRA3 findings the We (16.09 macrophages these that controls cells. positive revealed healthy myeloid assay CD68 than Immunofluorescence in LP. in cells with the expressed expressed Compared in mainly were macrophages 1E). is the protein (Figure stain LILRA3 to LP study, with antibody the previous saturated LILRA3 to in and were form, According located biopsies soluble (0.21 1G). cells a UC CD of in and the exist cytoplasm normal. CD from to (0.16 the were reported controls the controls in is non-IBD of healthy LILRA3 LILRA3 the the (LP) 1E). found (Figure from propria consistently neutrophils mucosa we lamina and of cells and staining structure plasma H&E sub-mucosa and biopsies, lymphocytes, intestinal the morphology in contrast, The LILRA3 of applied. In expression were situ in IHC and qRT-PCRand changes the pathological detect with further accordance To in (2.50 CD and patient both extracted, in the was 1D). expression (Figure in samples LILRA3 in UC observed increase 10 were noteworthy (2.26 and levels a showed CD mRNA blotting 7 western LILRA3 data, non-IBD, Increased controls, 8 healthy included. (p 14 another were controls. of from healthy total samples with a UC Overall, compared carried 52 analysis. samples groups that then intestinal this and finding we genotype, from CD our deletion excluded gut, on the directly 36 the for were Based involves homozygous LILRA3 expression. are undetectable primarily LILRA3 expression with intestinal that LILRA3 investigate undetectable disease to with inflammatory blotting samples western chronic and a qRT-PCR is out found IBD we deletion that excluded, were the Considering (2.11 subjects for CD (0.98 deletion the controls heterozygous both homozygous the in those the mRNA with the LILRA3 when compared in of for addition, levels increased homozygous the In in subjects significantly gene. increases in remarkable was wild-type CD, undetectable available with the expression with almost patients with samples was its 36 from and LILRA3 derived whereas for 1A, were assessed deletion, Figure enrolled was in subjects 6.7-kb blood The shown controls. peripheral As healthy in data. 53 genotyping expression and UC its with on controls patients variation healthy and 48 LILRA3 with patients of compared UC effect expression the The stratified. LILRA3 after between increased or frequencies whole exhibit allele a patients and as IBD groups genotypes complications the the penetrating for or for observed stricturing was association intestinal patients controls develop no CD the to However, that likely revealing 2). less analyzed, (Table were then genotype were controls rs410852 p the the (p=0.04, and carrying groups CD patient for of phenotypes locus the risk between a be to found ± .2 ru ncmaio ihhatycnrl (0.39 controls healthy with comparison in group 1.72) ± .3 n Cptet (0.19 patients UC and 0.03) ± .0) aymr IR3pstv el eedtce nteL fsamples of LP the in detected were cells LILRA3-positive more many 0.009), ± ± .9 p005frC;p004frU)(iue1B). (Figure UC) for p=0.014 CD; for (p=0.005 0.59) .3frC,6.57 CD, for 5.03 FDR < .0 o ohC n Cgop Fgr C.Ttlprotein Total 1C). (Figure group) UC and CD both for 0.001 01,O=.2 5C=.117)(al ) Relationships 1). (Table 95%CI=1.01-1.73) OR=1.32, =0.12, ± .3 (p 0.03) ± 6 .6frH,p HC, for 1.96 γ (p < ± γ, < .7 (p 0.27) .1frC;p CD; for 0.01 TNF- .0) TNF- 0.001), < α < ± swl sI-.Clsclue for cultured Cells IL-6. as well as .1 Fgr A2) nsummary, In 2A-2C). (Figure 0.01) .7 n C(1.72 UC and 1.47) .5frbt DadU group) UC and CD both for 0.05 α < (p .5frU)(iue1E, (Figure UC) for 0.05 < .0) n L6(p IL-6 and 0.001), α ± ± downregulates .0 patients 1.10) .8 n UC and 1.68) + LILRA3 < + Posted on Authorea 15 Apr 2020 — CC BY 4.0 — https://doi.org/10.22541/au.158696001.15833660 — This a preprint and has not been peer reviewed. Data may be preliminary. n h ieec a ut infiata 8ad7 or p006fr4 or;p003fr7 hours) found 72 we for phosphorylated and pathway, p=0.003 of hours; signaling levels 48 vector, potential was the for proliferation the null increased (p=0.016 enhanced investigate the significantly hours culturing, to LILRA3 expressing MAPK 72 of applied of and cells hours was overexpression 48 24 the blotting that at After with Western significant hours. compared 5A). quite 72 was (Figure cells and difference 48 U937 the 24, LILRA3-overexpressing and 12, detecting the by at assay for CCK-8 nm using evaluated observed 450 was lines at cell values U937 established MEK/Erk OD two and the of Akt capacity of proliferation The combination a expression. through CD36 cells increasing U937 pathways by of signaling cells proliferation U937 the of enhances ability LILRA3 phagocytosis the of enhances phagocytosis ability LILRA3 the phagocytosis that and (p antibody reveal the manner lines, specific decrease dependent cell CD36 significantly concentration p LILRA3-overexpressing could 4E). a ture, of antibody 4D, at significantly culture lines specific 4C, could the cell The (Figure LILRA3 LILRA3-overexpression to again. CD206 that added detected on membrane found was was influence We plasma efficiency USA) no the 2). Jose, Table had on San Supplementary yet receptors two (BD, see CD36 these of reaction, of expression the expression promote in alter used phagocy- could primers LILRA3 in (for whether roles explore monocytes important further on play to vector expressed (SR), null receptors receptor the endocytic scavenger effective expressing and cells (MR) CD206/ with receptor tosis. percenta- comparison mannose the in including analyzing hours mainly by 6 30.03% hours at hours; 24 beginning 12 and beads 12 engulf 6, to (5.23% 0.5, ability at strong FCM a by GFP detected expression of was CD36 ge upregulating efficiency suppressing by phagocytosis by cells The mainly U937 lines of cell phagocytosis U937 promotes of LILRA3 capacity migration the (p attenuates IL-8 exogenous secretion. LILRA3 showed the IL-8 results that by assay reversed suggest migration by was the data LILRA3, and suppressed by The chamber, significantly inhibited upper capacity, the were migration to (CCL2, the USA) CXCL10 that Hill, chemokines IL-8, Rocky (Peprotech, CC-type CCL3, CXCL8 two or CCL2, CCL2 of that expressed predominantly expression found (p were detect we LILRA3 which Interestingly, CXCL10), to (CXCL8/IL-8, monocytes. assay chemokines in CXC-type in Elisa two incubation other performed of directing and physiologically then (p hours CCL3) lower mediators We the key vector 6 into as migration. null after recognized migrated cell are the that had receptors LILRA3 found harboring coupled overexpressing We G-protein cells cells seven-transmembrane, migration. U937 with the cell compared of investigate few chamber to RPMI-1640, chemokines containing applied certain FBS was of 10% test expression transwell decreasing by A migration cell U937 attenuates LILRA3 rate cells. U937 apoptosis (p late in the frequencies apoptosis decreased regulating apoptosis obviously (3.64% total LILRA3 GFP cells and although vector for that apoptosis harvested null calculated conclude were the was cells we hours, with 3E, ratio 48 and compared apoptosis for 3D the culturing Figure After and in kit, reported. shown 7-AAD been apoptosis not with cell has stained apoptosis U937 and on on LILRA3 of effects impact limited The has LILRA3 of presence The molecule. anti-inflammatory an as inflammation in < ± Fgr B5) ie htteMKadAtptwy r lsl eae opoieain our proliferation, to related closely are pathways Akt and MEK the that Given 5B-5D). (Figure .1fr1gm n p and 1mg/ml for 0.01 0.21% + < ea Red Texas MRC1 .0)(iue3) ovrf u nig,w hnaddacranaon frecombinant of amount certain a added then we findings, our verify To 3G). (Figure 0.001) vs. ± 0.83% 3.80% a motn R n CD36/ and MR) important (an + ± vs. el.A hw nFgr Aad4,LLA-vrxrsigU3 el showed cells U937 LILRA3-overexpressing 4B, and 4A Figure in shown As cells. .5,p 0.35%, 26.43% < .0 o 0gm t2hclue Fgr F.Tkntgte,orresults our together, Taken 4F). (Figure culture) 24h at 10mg/ml for 0.001 ± < .4,p 0.64%, .1a or;26.00% hours; 6 at 0.01 ± 0.11% > < .5.Ti eutidctsta IR3i o novdin involved not is LILRA3 that indicates result This 0.05). .1a 4hus.Ptenrcgiinrcpos(PRRs), receptors recognition Pattern hours). 24 at 0.01 vs. 7 [31,32] SCARB3 4.41% R-C n etr ltigwr employed were blotting western and qRT-PCR . ± < .0,p 0.20%, .0)(iue3) hmknsadtheir and Chemokines 3F). (Figure 0.001) a motn ls R r w highly two are SR) B class important (an ± 0.17% < vs. < .1,i a iteeeto early on effect little had it 0.01), .1frbt rusa 2 cul- 12h at groups both for 0.01 + 20.47% el.Acrigt h data the to According cells. ± Akt .2,p 0.42%, < .1 Fgr 3H). (Figure 0.01) , MEK < and .0 at 0.001 P38 Posted on Authorea 15 Apr 2020 — CC BY 4.0 — https://doi.org/10.22541/au.158696001.15833660 — This a preprint and has not been peer reviewed. Data may be preliminary. IR3sossrn n pcfi idn otesraeo rmr eihrlbodmnncercells mononuclear blood peripheral primary of surface TNF- the (LPS)-induced to mono-myeloid cells lipopolysaccharide responses B binding on and immune cells co-expressed specific monocyte innate primarily U937 and (PBMCs), receptors regulating strong of as shows family recognized system. LILRA3 homologous immune increasingly highly in is the roles leukocytes, of certain Based play member LILRB1. might a and LILRA3 LILRB2 LILRA3, that of functions speculate with affect we disequilibrium might findings, linkage LILRA3 these strong that in on reported were study alleles Previous LILRA3 alleles. of additionally, LILRB2 LILRB2, members regulate to and two hence LILRB1 are HLA-I to with LILRB2 homologous molecular and CD8 expression LILRB1 of combination LILRA3 controls. the healthy found block CD8 to in we reported Unexpectedly, than was LILRB1 higher CHB. family. significantly with LILRB was compared susceptibility. patients China IBD IBD Central in of and studies population LILRA3 polymorphism previous undetectable Han in with among the LILRA3 results consistent and deletion between is rs410852 6.7-kb which homozygous rs103294, association that between levels, demonstrated observed we the was genotypes, development deletion IBD clarify 6.7-kb with to association significant was no Although goal initial unknown or Our above the of one with Discussion interacting by pathway signaling 7). above (Figure the receptors activate might LILRA3 that antigen- leukocyte molecule human of HLA-I classical The non-classical unknown. the remains and LILRA3 LILRA3 for for receptor pathway exact signaling proliferation, The cell possible regulates the LILRA3 of that pathways. schematic indicate signaling A PI3K/MEK/Erk data are and our investigations that PI3K/Akt conclusion, further is through In phosphorylation; phenomenon likely Foxo3 speculation. this most on a this explain effect detected verify to inhibitory we to hypothesis direct/indirect contrast, needed possible in a A have MEK; overexpressed. might and finding was our Akt LILRA3 cells. LILRA3 activated with vector accordance by when null in enhanced level critical and not decreased LILRA3-overexpressing be well-known was between should result phosphorylation a detected phosphorylation this was in Foxo3a is Foxo3a inhibitors, decline Indeed, Foxo3a phosphorylated three noteworthy of the a level with speculation. the detected pretreated when our we cells in with Although with protein compared growth. consistent this proliferation cell of were cell Most in on results involved decreased 6D). efficiency markedly These factor (Figure also inhibitory transcription dramatically PI3K) highest (DMSO) inhibitors. of the could sulfoxide inhibitor other with inhibitors dimethyl specific viability MEK, the (a three with and cell LY294002 the treated 6D, Akt reduce and that cells of sharply carried 6C phosphorylation found with Figure could was We compared in and assay shown culture viability. 6A-6C) as CCK-8 importantly, of cell (Figure the hours detect proteins and 24 to targeted efficiency, after hours their inhibitory 72 of the and phosphorylation evaluate decrease 48 to 24, to used at synergistically out was function blotting Western might and 1 6- pathways USA). (GSK1120212, Akt into MEK two seeded of both were inhibitors the Because cells specific μ LILRA3-overexpressing that with 2). hypothesis, pretreated speculated Figure our and confirm plates we Supplementary To well PI3K, (see proliferation. of cell by found U937 Phosphorylation activated was regulate 5G). be difference and can no 5F decreased 5E, Foxo3a but MEK of 5C, investigated, that (Figure but also LILRA3 increased was significantly of was presence PI3K and the c-Raf in PDK1, Erk, kinases. of two phosphorylation these that of respectively), factors MEK, downstream and and and Akt upstream for the kinases exploring phosphorylated of on levels The focused mainly research ensuing o/)o IK(Y902 0mo/) h he niioswr ucae rmSlekhm(Houston, Selleckchem from purchased were inhibitors three the mmol/L); 20 (LY294002, PI3K or mol/L) ooA Nogo Foxo3a + el.LLB rmtdhmtpitcse el(S)poieain osdrn htLLA is LILRA3 that Considering proliferation. (HSC) cell stem hematopoietic promoted LILRB2 cells. T r eotda osbercposfrLILRA3 for receptors possible as reported are ) tesae ontempoeno E n k)wr ute xmnd nrgigy efound we Intriguingly, examined. further were Akt) and MEK of protein downstream shared (the Erk teacpe ontempoeno MEK), of protein downstream accepted (the HLA-G α PI3K [23] erto ymncts n nrae nLLA aebe de- been have LILRA3 in increases and monocytes, by secretion diinly eobnn IR3cuddaaial abrogate dramatically could LILRA3 recombinant Additionally, . [18,19] swl as well as tecnetoa ptempoeno ohAtadMEK), and Akt both of protein upstream conventional (the nadto,w eetdamc ihrdlto ratio deletion higher much a detected we addition, In . 8 oo66 Nogo [22,33,34] ae nteefidns ehypothesize we findings, these on Based . ahgl osre 6aioai loop acid 66-amino conserved highly (a μ o/) k M-262C,5 2HCL, (MK-2206 Akt mol/L), PDK1 P65 [24,35] , SAPK/JNK nvto recombinant vitro, In . and c-Raf (HLA-I): I teclassical (the and HLA-C mTOR Posted on Authorea 15 Apr 2020 — CC BY 4.0 — https://doi.org/10.22541/au.158696001.15833660 — This a preprint and has not been peer reviewed. Data may be preliminary. etFx3 hshrlto hog nukonmcaim ute xeiet r eddt elucidate to af- needed directly/indirectly are might experiments LILRA3 Further that mechanism. is unknown mechanism. inconsistency an this this through for phosphorylation explanation Foxo3a MEK, possible fect of One inhibitors PI3K. was specific Foxo3a with and of pre-incubated Akt were phosphorylation cells contrast, LILRA3-overexpressing In when overexpressed. reduced to was conspicuously degradation nucleus LILRA3 when the MDM2-mediated decreased from was via phosphorylation translocation Foxo3a activity by inhibiting transcriptional followed by of protein, growth inhibition 14-3-3 the with to phosphorylation cytoplasm, bind specific the via to Foxo3a Foxo3a of causes apoptosis activity phosphorylation arrest, transcriptional cycle the to cell regulate known tumorigenesis, to is proliferation, factor, metabolism, transcriptional longevity as tumor-suppressive two such and a events are Foxo3a, cellular signaling proliferation. various growth PI3K/MEK/Erk CD8 cell regulate and and regulate of survival PI3K/Akt to proliferation cell pathways vitro. (MLR) two in the in reactions involved proliferation induce pathways lymphocyte cells could essential U937 mixed LILRA3 enhance in recombinant could cells pression that with NK reporting consistent and are studies T-cells results previous These expression. to CD206 Similar on effect divided limited assay. further a phagocytosis had be our but lines can expression cell B CD36 platelets, cultured class Mø, increased DCs monocytes, of and and as variety Mø A such by lineages, ligands a Class expressed SRBIII hematopoietic many and B. and and lymphoid with class cells SR-BII, of cells receptors SR-BI, and endothelial on SR-AIII, of A expressed mainly SR-AII, class family is SR-AI, is subgroups: CD36, a including two Phagocytosis of types into cells. many consists membrane divided into U937 SR phagocyte be of endocytosis. the can vector. capacity with on null and phagocytosis associated present the the receptors receptors harboring promote cells important endocytic can than by LILRA3 beads mediated that latex predominantly more indicates from engulfed result migration cells This LILRA3-overexpressing monocyte study, prevent agreement this might was inflammation. in In LILRA3 LILRA3 suppressing is when that thereby finding cells tissues, speculate by This U937 into we attenuated of cells. circulation results, capacity U937 the sharply these migration on was on impaired LILRA3 molecules Based demonstrated cells and these overexpressed. which monocytes, assay, of U937 by transwell expression secreted of our chemokines reduce ability with main conspicuously the and migration chemokines to are between Mø able the CXCL10 interaction into was and by that CXCL8 blood differentiate mediated CCL3, found the to largely CCL2, We is LILRA3. through sites receptors. migration circulate infected vivo, chemokine cells or In and responses. These tissue inflammatory damaged boost body. responses to immune to human recruited adaptive in triggering are thus leukocytes and DCs, all system of lymphatic responses. 2–10% and immune for in our LILRA3 account observed in of we responses, Monocytes effect and immune anti-inflammatory on cells, an LILRA3-overexpressing LILRA3 confirms IFN- further of establish decrease finding effect to markedly critical the employed could are LILRA3 deduce were LILRA3 that To that monocytes monocytes mainly speculate We and U937 monocytes. to pathogenesis. disorder human on reported IBD study, immunologic function in was by certain roles LILRA3 important characterized exert played and might is and circulation, responses IBD on immune in Since CD68 in expressed monocytes regulators more mainly from possessed monocytes. is patients derived on LILRA3 CD that mainly expressed and found are intestinal also of macrophages we LP mucosal nal study, in the our role diseases. in essential inflammatory In located an in conventionalmacrophages play inflammation. including LILRA3 collectively and subsets, for that (MNP) infection role macrophages phagocyte critical tissue-resident homeostasis, mononuclear and a of monocytes indicate array cells, data diverse dendritic these a contains All LP patients. intestinal SLE The and MS in tected [42-44] rvossuishv hw httetokyknssAtadEkhv h ability the have Erk and Akt kinases key two the that shown have studies Previous . [32] norsuy ohC3 n D0 eedtce nU3 el,adLILRA3 and cells, U937 on detected were CD206 and CD36 both study, our In . γ, TNF- [36] hrfr,siuaino ooyemgaincnhelp can migration monocyte of stimulation Therefore, . α [38,39] 9 n L6sceinadices L1 erto.This secretion. IL-10 increase and secretion IL-6 and [40,41] [43,44,48] Ri abhdaebnigrcpo mainly receptor carbohydrate-binding a is MR . n efudta IR3atvtdthese activated LILRA3 that found we and , [21] [44] eosre hticesdLLA ex- LILRA3 increased that observed we , h a/r aha lorgltscell regulates also pathway Ras/Erk The . norsuy eosre htFoxo3a that observed we study, our In . [45-47] [29,37] k-o MEK-mediated or Akt- . + LILRA3 RII lotermed also SRBIII, . [37] RadM are MR and SR . + el.Intesti- cells. + Posted on Authorea 15 Apr 2020 — CC BY 4.0 — https://doi.org/10.22541/au.158696001.15833660 — This a preprint and has not been peer reviewed. Data may be preliminary. ietasrp/ilrcl niioyrcpo oii h ekct eetrcomplex. receptor leukocyte the 3959-3967. in p. loci 28(12): receptor 1998. munology, inhibitory Wilson, M.J. cell and Trowsdale, transcript/killer J. like Colonna, M. Norgate, Z. M., deletion. Torkar, 9. 6.7-Kbp a from results gene 3655-3662. Wilson, ILT6 p. M.J. the and of Beck, S. allele Milne, null S. Haude, Wilson, A. M.J. M., Torkar, and 8. Trowsdale, J. Haude, A. receptors. Torkar, NK-cell M. R., Barten, 7. Andreas, Z. and Katja, complex . 4778-4783. L. receptor Hagen, leukocyte p. Trowsdale, W. 97(9): J. V., 2000. Armin, and America, Beck, 6. of families. S. States gene Sheer, United KIR/ILT D. the Jones, human of T. of Sciences Milne, sequences S. and Haude, organization A. Torkar, the M. M.J., research. Wilson, of 5. years 55 of Zhi, R.G. meta-analysis and 161-166. A Xiao, H.S. China: Zhao, mainland H. Teo, Xia, in E.K. S.Z. Ju, and J., Zheng, Ng, 4. T.M. Singapore. See, in disease S.J. 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B., Khor, 1. authors. the of References any for declare to interest of conflict financial no is There number study. statement (grant this interest China in of participate of Conflicts who Foundation individuals Science all thank Natural authors National The from grants by 81800488). supported 81470823, was work This to cell immunity. needed patients. regulate adaptive are IBD efforts in and Acknowledgments Further in LILRA3 pathways. responses of increased signaling immune role MEK/Erk markedly innate exact and is in the Akt expression of explore modulator combination LILRA3 anti-inflammatory a that an through proliferation as report we function might time, LILRA3 first the 3) for immunity; Collectively, adaptive the increased microorganisms. of CD8 LILRA3 ability and activation 4) monocytes, pathogens migration circulation; restricting as more in their kill such thereby pathogens more immunocytes, attenuating tissues, cells engulf of by in to U937 proliferation cells system DCs monocytic in enabled and circulation molecule LILRA3 cytokine Mø in increased proinflammatory anti-inflammatory reduced decreasing monocytes an by accordingly restrained inflammation as and inhibited LILRA3 function directly 2) LILRA3 might 1) secretion; LILRA3 following: the that on found based we together, Taken rnsi muooy 01 21:p 52-57. p. 22(1): 2001. Immunology, in Trends muooia eiw,20.111:p 39-51. p. 181(1): 2002. Reviews, Immunological namtr oe ies nteAi–aicae:Acmaio with comparison A area: Asia–Pacific the in disease bowel Inflammatory ora fGsretrlg eaooy 00 56:p 622-625. p. 15(6): 2000. Hepatology, & Gastroenterology of Journal eeisadptoeei fiflmaoybwldisease. bowel inflammatory of pathogenesis and Genetics ora fDgsieDsae,21.11. 2010. Diseases, Digestive of Journal eei fteITLRMRcutr ihntehuman the within clusters ILT/LIR/MIR the of Genesis 10 + -el n Kcls hsalwn hs el to cells these allowing thus cells, NK and T-cells rvlneadicdnertso rh’ disease Crohn’s of rates incidence and Prevalence aildffrnei h rvlneo ulcerative of prevalence the in difference Racial ragmn fteITgn lse:Acommon A cluster: gene ILT the of Arrangement uoenJunlo muooy 01 30(12): 2001. Immunology, of Journal European ora fDgsieDsae,21.1() p. 11(3): 2010. Diseases, Digestive of Journal rceig fteNtoa cdm of Academy National the of Proceedings stpcvraino oe immunoglobulin- novel of variation Isotypic iegn n ovreteouinof evolution convergent and Divergent uoenJunlo Im- of Journal European lsiiyin Plasticity Nature, Posted on Authorea 15 Apr 2020 — CC BY 4.0 — https://doi.org/10.22541/au.158696001.15833660 — This a preprint and has not been peer reviewed. Data may be preliminary. 3 e,THY,A icel .LuLu .A,P aesaih .Wsne,M atr,K rat and Bryant, K. Raftery, M. Wasinger, Protein. V. 32873-32885. Rajeaskariah, A3 p. Receptor P. Geczy, Immunoglobulin-like An, C.L. Leukocyte H. Active Klotzsch, Lau, Liu E. S. Tedla, Lord, Mitchell, N. M.S. A. Guillemin, T.H.Y., Lee, G.J. 23. Lim, C.K. Heng, 66. B. Nogo Lee, with T. Brettle, al., M. et H., al., An, et Immunostimulatory Torsten, the 22. M. of E., Characterization S.R. Functional Renata, and S. Gamze, B-NHL K. with Dietrich, Molecule. Deletion P. LILRA3 Nadine, interferon-gamma. D. the Michael, Tedla, of and T. Association N. arthritis Sandra, factor-alpha, R. and rheumatoid necrosis L.H., Bryant, Zhi, with tumor 21. K. patients 10, 1596-1606. Mcneil, in interleukin p. H.P. increased 37(8): Geczy, by 2010. is regulated C. Rheumatology, protein, Borges, tightly antiinflammatory L. is natural Piraino, and potential B. Serge, Chandra, a N. with V. Associated LILRA3, and Not H., Is Nicodemus, An, Deletion T. 20. Gene Katherine, LILRA3 and 6.7kbp B. Sclerosis Severity; William, Multiple Disease Susceptibility. with R. of Diseases Indicator Patients Ute, Independent in Strong V.-C. Increased a Is J., Is (LILRA3) G.G. A3 Receptor Chai, Immunoglobulin-Like L. Leukocyte syndrome. Sjogren’s H., and 2070-2075. An, p. erythematosus 19. 74(11): lupus 2014. systemic Diseases, Li, of Z. Rheumatic subphenotypes and the Yang, of and Y. susceptibility He, on J. Su, RA3) McNeil, to Y. H.P. correlations Y., and Du, synovium: Geczy, 18. rheumatoid C. Bryant, in receptors K. Vollmer-Conna, immunoglobulin-like activity. 19q13.4. U. leukocyte disease and Borges, inhibitory L. 9q31.2 and An, at activating H. loci al., et N., risk Shao, Tedla, cancer Q. Wang, 17. prostate X. new Xu, 1231-1235. C. two Jin, p. identifies G. 44(11): Liu, men 2012. F. Wang, Chinese M. Ye, in D. study Mo, Z. al., J., et Xu, L, 16. Siqun Z. Jian, K. 8832-8840. Population. Zhuo, p. Chinese C. a Rong, 14(5): N. in 2013. Lu, Risk T. Hyperplasia Sha, Prostatic T. Benign Xin, with G. Li, W. J., Yang, 15. M N. Epplen, 445-447. J.T. Nigmatova, al., V. et Goedde, Schmidt, R. Rheumatoid Antibody-Positive S., Protein Koch, Anti-Citrullinated 14. of Severity and Susceptibility Li, in Z. Arthritis. and Bias Hu, Sex F. to Liu, X. RA3, Cui, Y. Y., Du, 13. 579-585. E. p. Moraru, 10(6): 2009. M. Immunity, Romo, & N. Ramil, al., L´opez-Botet, et M. E. Mart´ınez-Rodr´ıguez,Munteis, Cisneros, S´anchez, J.E. E. A.J. Ord´o?Ez, D., Witte, 12. T. and Sj Reuter, with S. deficiency Matthias, 6 T. 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Antigens, Tissue OOfcos atro ieaddeath. and life of matter a factors: FOXO 00 2(96:p 656-661. p. 327(5966): 2010. soito ewe GRplmrhssadters fln cancer. lung of risk the and polymorphisms EGFR between Association L ls lei euneadcnomto euaeluoyeIg-like leukocyte regulate conformation and sequence allelic I class HLA ua niioyrcposI-ietasrp IT)adILT4 and (ILT2) 2 transcript Ig-like receptors inhibitory Human ora fCiia netgto,19.90. 1992. Investigation, Clinical of Journal muooyLtes 05 6() .343-348. p. 168(2): 2015. Letters, Immunology h IRfml:mdltr fint n dpieimmune adaptive and innate of modulators family: LILR The ora fInt muiy 04 () .727-738. p. 6(6): 2014. Immunity, Innate of Journal nixdRdxSga,20.756:p 752-760. p. 7(5-6): 2005. Signal, Redox Antioxid lo ooye:Dvlpet eeoeet,adRela- and Heterogeneity, Development, Monocytes: Blood ora fLuoyeBooy 02 92. 2012. Biology, Leukocyte of Journal teoceoi,20.121:p 1-15. p. 182(1): 2005. Atherosclerosis, 12 D6 ls cvne eetrivle nangio- in involved receptor scavenger B class a CD36: emnto fImn ciain nEssential An Activation: Immune of Termination ora fCiia netgto,20.108(6): 2001. Investigation, Clinical of Journal OOTasrpinFcosi Cell-Cycle in Factors Transcription FOXO uueOclg,20.21:p. 2(1): 2006. Oncology, Future adoaclrRsac,2015. Research, Cardiovascular rceig fteNational the of Proceedings eeomn fMonocytes, of Development ebaeGlycoprotein Membrane uoenJunlof Journal European Biochemistry M- gene MMP-9 lo,1992. Blood, Alternative Posted on Authorea 15 Apr 2020 — CC BY 4.0 — https://doi.org/10.22541/au.158696001.15833660 — This a preprint and has not been peer reviewed. Data may be preliminary. 6(48246.)252(65.3)252(65.3) A* G lele Al- AA* AG GG, rs410852 C* T lele Al- CC* CT 660(64.8)244(65.9) TT, rs103294 +* - Allele +/+ /+ -/-,- tion dele- kb 6.7 al :AscainbtenLLA oyopimadIDdevelopment IBD Name and polymorphism Blenis, LILRA3 J. between Arden, Association K.C. Anderson, 1: M.J. Table Hu, 857-868. L.S. Juo, p. P. 96(6): Lin, M.Z. 1999. 1978-1986. Zigmond, M.J. p. al., Bonni, 1813(11): et A. 2011. A., Research, Brunet, Rishi, Cell A.K. 48. Molecular and - Hadden, (BBA) T.J. Acta Tang, Biophysica N. et X., Zhang, 47. 5392(282). C., H, M 46. 2(10). 2008. biology, cell J., Yang, 45. K., 6728(398). J, G 44. AFX. Rena, and G. FKHRL1 and Y.L. Woods, 352-360. 43. p. Kops, 27(7): G.J.P.L. 2002. and Sciences, B.M.T. Biochemical Burgering, 42. c 91.)1(.)02 .11.39(0.78- 0.41 0.27 16(8.6) 59(11.6) * k rmtsCl uvvlb hshrltn n niiigaFrha rncito Factor. Transcription Forkhead a Inhibiting and Phosphorylating by Survival Cell Promotes Akt 5(84199.)175(90.7) 154(15.1) 864(84.9) 14(2.8) 495(97.2) 322(31.6) 696(68.4) 56(11.0) 453(89.0) 358(35.2) 450(88.4)169(91.4) (%) N (N=509) a HC euaino eldahpoes aps- yphosphorylation. by caspase-9 protease death cell of Regulation R rmtstmrgnssb niiigFX3 i D2mdae degradation. MDM2-mediated via FOXO3a inhibiting by tumorigenesis promotes ERK ietcnrlo h okedtasrpinfco F ypoenkns B. kinase protein by AFX factor transcription Forkhead the of control Direct 55(14.9) 315(85.1) 9(4.9) 176(95.1) 96(25.9) 274(74.1) 20(10.8) 165(89.2) 126(34.1) (%) N (N=185) CD iceia oit rnatos 02 04:p 391. p. 30(4): 2002. transactions, Society Biochemical 0.90 0.17 0.04 0.94 0.70 P (N=185) CD ffc fmlil hshrlto vnso h rncito atr FKHR, factors transcription the on events phosphorylation multiple of Effect 0.90 0.40 0.12 0.94 0.90 b (N=185) CD P FDR elcceaddahcnrl oglv Forkheads. live long control: death and cycle Cell 1.43) 1.02(0.73- 1.30) 0.55(0.24- 1.73) 1.32(1.01- 1.75) 1.02(0.59- 1.35) 1.05(0.82- 2.47) (95%CI) OR (N=185) CD 13 k,Fx n euaino apoptosis. of regulation and FoxO Akt, 50(13.0) 336(87.0) 6(3.1) 187(96.9) 129(33.4) 257(66.6) 21(10.9) 172(89.1) 134(34.7) 893 893 .909 .61.28(0.73- 0.96 0.96 0.39 18(9.3) 18(9.3) (%) N (N=193) UC cec NwYr,NY) 1998. N.Y.), York, (New Science 50(13.0) 336(87.0) 6(3.1) 187(96.9) 129(33.4) 257(66.6) 21(10.9) 172(89.1) 134(34.7) (%) N (N=193) UC 0.30 0.80 0.52 0.96 0.87 P P (N=193) UC aue 1999. Nature, Biochimica rnsin Trends Nature 0.78 0.96 0.78 0.96 0.87 (N=193) UC Cell, FDR 0.78 0.96 0.78 0.96 0.87 P (N=193) UC FDR 1.69) 1.20(0.85- 2.33) 0.88(0.33- 1.18) 0.92(0.72- 1.72) 1.01(0.60- 1.30) 1.02(0.80- (95%CI) OR 2.22) (N=193) UC 1.69) 1.20(0.85- 2.33) 0.88(0.33- 1.18) 0.92(0.72- 1.72) 1.01(0.60- 1.30) 1.02(0.80- (95%CI) OR 2.22) 1.28(0.73- (N=193) UC Posted on Authorea 15 Apr 2020 — CC BY 4.0 — https://doi.org/10.22541/au.158696001.15833660 — This a preprint and has not been peer reviewed. Data may be preliminary. al :AscainbtenLLA oyopimadIDi upeoyecnrlcohort subphenotype-control in IBD and polymorphism LILRA3 between Association 2: Table c Phenotype6.7 b a < e age Diagnosis (N) CD (N) d 40 17- ior hav- be- Disease f per Up- L4 tis i- col- Ileo- L3 Colonic L2 Ileal L1 ease dis- of Site > GI :rpeet h idgntp rwl allele. wild or genotype wild the represents *: P (yr) C elh controls healthy HC: HC 17 03 .01.11(0.38- 1.00 4 34 40 FDR deletion kb +/+ VS /+ ,- /- - 6 6152 01699 9 176 Ref Ref Ref 14 14 495 Ref 20 Ref Ref 20 Ref 165 56 56 0.79(0.19- 453 1.00 2 Ref 13 16 Ref 169 59 450 2 01.60(0.80- 10 122 4 69 51 45 ersnsthe represents : +/+ VS /+ ,- /- - deletion kb 6.7 0 5 8 3 P deletion kb 6.7 1.00 0.21 0.66 0.26 P au dutdb as icvr aecorrection. rate discovery false by adjusted value R9%I TT,CT OR(95%CI) deletion kb 6.7 3.87) 3.22) 1.17) 1.13(1.10- 4.67) 1.81(0.70- 1.85) 0.84(0.38- 6.53) 1.97(0.59- 3.25) CC VS rs410852rs410852rs410852rs410852rs410852rs410852rs410852rs410852rs103294rs103294rs103294rs103294rs103294rs103294 22210 .00.74(0.16- 1.00 1.00 2 2 12 1 31 .308 1.08(0.57- 0.83 0.83 13 13 113 4 0.99(0.38- 67 1.00 51 1.00 43 5 5 40 CC VS TT,CT 0 7 8 5 14 P P 0 7 8 5 1.00 0.69 0.56 0.90 R9%IO (5C)GG,AG OR(95%CI)OR(95%CI) 1.00 0.69 0.56 0.90 3.40) 2.03) 1.16) 1.12(1.09- 2.70) 1.18(0.52- 1.75) 0.79(0.36- 2.80) 1.06(0.40- 2.61) 3.40) 0.74(0.16- 2.03) 1.08(0.57- 1.16) 1.12(1.09- 2.70) 1.18(0.52- 1.75) 0.79(0.36- 2.80) 1.06(0.40- 2.61) 0.99(0.38- AA VS AA VS GG,AG 40010 .01.03(1.01- 1.00 1.00 0 0 14 2 .803 0.57(0.21- 0.38 0.38 6 6 120 4 0.40(0.11- 71 0.31 56 0.31 45 3 3 42 AA VS GG,AG 0 3 3 3 P P 0 3 3 3 1.00 0.80 0.55 0.36 OR(95%CI)OR(95%CI) 1.00 0.80 0.55 0.36 1.04) 1.50) 1.04) 1.03(1.01- 2.39) 0.67(0.19- 1.89) 0.53(0.15- 1.53) 0.42(0.12- 1.43) Posted on Authorea 15 Apr 2020 — CC BY 4.0 — https://doi.org/10.22541/au.158696001.15833660 — This a preprint and has not been peer reviewed. Data may be preliminary. h .-bdlto nLLA RAepeso nprpea lo.mN xrsinwseautdby evaluated was expression mRNA controls. blood. healthy peripheral with in compared expression mRNA patients LILRA3 IBD on in deletion increased 6.7-kb is the LILRA3 1: Figure Legends Figure f e d Extensive E3 sided Left- E2 tis ti- Proc- E1 tion ca- lo- ease Dis- > 40 17- < (yr) age Diagnosis (N) UC ease dis- anal ri- Pe- ing trat- e- Pen- B3 ing tur- Stric- B2 penetrating Non- stricturing/ Non- B1 I gastrointestinal GI: r year yr: C elh controls healthy HC: 40 17 86 70 19 76 96 3 175 26 22 33 114 6 8 4 10 8 0 18 1 1 4 11 0.15 0.73 0.61 0.99 0.25 1.00 0.34 0.46 1.00 0.37 4.49) 1.88(0.79- 2.50) 1.15(0.53- 1.89) 0.62(0.21- 2.03) 1.00(0.49- 3.40) 1.57(0.73- 1.17) 1.13(1.10- 25.59) 3.41(0.45- 21.79) 2.88(0.38- 3.16) 1.08(0.37- 2.67) 1.36(0.69- 85 68 19 77 73 22 172 25 21 31 113 7 10 4 11 10 0 21 2 2 6 12 15 7 10 4 11 10 0 21 2 2 6 12 0.33 0.64 0.54 0.68 0.78 0.20 0.79 0.99 0.49 0.65 0.33 0.64 0.54 0.68 0.78 0.20 0.79 0.99 0.49 0.65 3.41) (0.62- 1.50 1.73) (0.41- 0.84 1.79) (0.19- 0.59 1.73) (0.43- 0.87 1.85) (0.44- 0.90 1.16) (1.09- 1.12 6.70) 1.55(0.36- 5.68) 1.30(0.30- 1.60) 0.64(0.26- 2.25) 1.16(0.60- 3.41) (0.62- 1.50 1.73) (0.41- 0.84 1.79) (0.19- 0.59 1.73) (0.43- 0.87 1.85) (0.44- 0.90 1.16) (1.09- 1.12 6.70) 1.55(0.36- 5.68) 1.30(0.30- 1.60) 0.64(0.26- 2.25) 1.16(0.60- A matof Impact (A) 89 76 22 84 101 2 187 26 21 33 122 3 2 1 4 2 0 6 1 3 4 3 3 2 1 4 2 0 6 1 3 4 3 1.00 1.00 0.49 0.57 0.90 1.00 0.54 0.04 0.03 1.00 1.00 1.00 0.49 0.57 0.90 1.00 0.54 0.04 0.03 1.00 2.98) (0.24- 0.84 4.82) (0.24- 1.08 4.95) (0.08- 0.62 1.85) (0.19- 0.59 6.38) (0.32- 1.43 1.04) (1.01- 1.03 5.81) 0.74(0.09- 0.74) 0.20(0.05- 0.75) 0.23(0.07- 4.07) 1.12(0.33- Posted on Authorea 15 Apr 2020 — CC BY 4.0 — https://doi.org/10.22541/au.158696001.15833660 — This a preprint and has not been peer reviewed. Data may be preliminary. sa.O t40n a eetda ,1,2,4 n 2hus B n )Wsenbo nlssof (NV). analysis cells PI3K, vector blot p-PI3K, Western null p-c-Raf, D) and times. PDK1, three and (OE) p-PDK1, least C of cells at analysis overexpressing (B, for blot repeated LILRA3 hours. (OE) Western was cells in 72 G) experiment overexpressing p-Foxo1 Each and and LILRA3 p-Foxo4, in F 48 Foxo3a, MAPK (E, 24, and P38 (NV). CCK-8 p-Foxo3a and 12, cells by MAPK 0, vector analyzed p-P38 null pathway at Erk, were and p-Erk, detected cells signaling MEK, U937 was p-MEK, possible vector nm Akt, the null p-Akt, 450 and and at cells proliferation OD U937 assay. cell LILRA3-overexpressing the U937 of increases Proliferation LILRA3 5: Figure el.GPHaudnewsue sacontrol. a as used was abundance GAPDH cells. times. three least at LILRA3-overexpression for repeated of was ability experiment phagocytosis Each The (F) manner. the and blotting. dependent decrease western concentration D 0.05, and significantly a (C, qRT-PCR at could to vector. lines according null CD36 cell expression the of CD206 expressing and antibody CD36 cells specific on U937 LILRA3 and of Effects cells E) U937 LILRA3-overexpressing between GFP of analyzed plasma percentage CD36 The upregulating hours. by 24 cells U937 of cell mean ability expression impaired phagocytosis as membrane the induced expressed enhances LILRA3 LILRA3 are and 4: Data Figure assay, times. Elisa migration three CCL2 IL-8. by least Recombinant the cells exogenous at (H) in U937 for the chamber LILRA3. were in repeated by upper by chamber secretion reversed the lower decreased chemokine was the to dramatically established on migration into added two were LILRA3 migrated were CXCL10 the of counts CXCL8 and in Impact Cell and IL-8 (%) (G) migration. CCL3, rate cytometry lines. cell CCL2, apoptotic cell U937 flow assay. of two on Representative analysis the LILRA3 and (D) Statistical between of cells (E) analyzed Effect U937 inhibition. LILRA3-overexpressing (F) vector. among of lines. null 7-AAD cell effect the and V-PE expressing significant annexin cells more with U937 a stained apoptosis showed IFN- of hours of analysis on 12 secretion LILRA3 and for of cells, Effect cultured U937 (B) IFN- the culture of blotting. to western Expression mechanisms. and mean related cells. Elisa as and qRT-PCR, U937 expressed by cells by cells U937 secretion established cytokine on two (C) LILRA3 the in of antibody. expression Effect primary the times. 3: three of Figure least instead at PBS for repeated with was treated ment mark were LILRA3 to Sections of used Quantification CD68 immunofluorescence. was for in antibody staining expressed monoclonal were CD68 protein CD68 CD68 Immunofluorescence. LILRA3 times. on by three expressed expression least macrophages. mainly LILRA3 at for of is repeated calization LILRA3 was experiment the 2: Each of Figure enrolled. were instead samples PBS columns) UC with two 14 and treated left *p CD were (the 14 Sections 30 NIBD, staining is 20 bar H&E immunohistochemistry. 3 Scale for (E) on antibody. staining control. 50 primary CD performed is control loading 7 were bar Negative (NIBD), a column) Scale (F) disease right expression as shown. bowel are (the Protein used non-inflammatory pictures LILRA3 (D) was representative 8 for excluded. from GAPDH immunohistochemistry LILRA3 extracted were samples. of and was levels UC expression Protein mRNA LILRA3 10 intestine. (C) undetectable the and excluded. in were possessing (-/-) LILRA3 Subjects deletion of intestine. the for the homozygous in samples when blood mean in the as expressed p are Data assay. qRT-PCR < < 0.01, ** + 0.05, LILRA3 p *** < ** 0.01, p p < < + 0.01, *** .0 esssm eoye nhatycnrl H) B RAepeso fLILRA3 of expression mRNA (B) (HC). controls healthy in genotypes same versus 0.001 el hnNnIDgop cl a s20 is bar Scale group. Non-IBD than cells ± p D C uent olce rmteLLA-vrxrsigU3 ellnswsused was lines cell U937 LILRA3-overexpressing the from collected Supernate (C) SD. < *** 0.001. p A ersnaieFMaayi fpaoyoi yU3 el t05 ,1 and 12 6, 0.5, at cells U937 by phagocytosis of analysis FCM Representative (A) . + < CD68 .0 essH rNB groups. NIBD or HC versus 0.001 * + + p μ .()Qatfiaino LILRA3 of Quantification (G) m. el nitsia ipis ahgopicue ape.Ec experi- Each samples. 5 includes group Each biopsies. intestinal in cells ea Red Texas < 0.05, ** * p p + < γ, < el a nlzd B hgctssrtswr statistically were rates Phagocytosis (B) analyzed. was cells 0.05, TNF- μ 0.01, ± o h etclm,30 column, left the for m 16 D apegop n eoye eeindicated. were genotypes and groups Sample SD. γ, * ** p *** α + p TNF- < swl sI- eesgicnl erae.Cells decreased. significantly were IL-6 as well as + p arpae nhmnintestinal. human in macrophages < arpae n Dptet osse more possessed patients CD and macrophages 0.05, < 0.01, α 0.001. μ o ahclm.()Ngtv control Negative (B) column. each for m n L6wsasse yEia aaare Data Elisa. by assessed was IL-6 and ** p *** + < p el nitsia ipis oa of total A biopsies. intestinal in cells 0.01, < 0.001. *** μ ± o h ih w columns. two right the for m p A eicto fLILRA3 of Verification (A) D aheprmn was experiment Each SD. < μ .0 essnl vector null versus 0.001 oo etos and sections, colon m A Lo- (A) (A) . * p ** < Posted on Authorea 15 Apr 2020 — CC BY 4.0 — https://doi.org/10.22541/au.158696001.15833660 — This a preprint and has not been peer reviewed. Data may be preliminary. in-ibd-patients-and-functions-as-an-anti-inflammatory-modulator investigated. Figures.doc regulate further to be signaling to needs PI3K/MEK/Erk file Foxo3a proliferation and Hosted Whether cell PI3K/Akt phosphorylation. LILRA3-induced Foxo3a trigger in inhibit role and directly/indirectly a might receptors plays LILRA3 LILRA3 unknown of proliferation. or pathway cell signaling listed U937 possible the the of of one schematic repeated A was 7: experiment Figure Each cells. U937 vector times. con- pretreated Null three a after DMSO. NV: (OE) as least time. cells used indicated at U937 was LILRA3-overexpressing for (A) abundance for of inhibitors for GAPDH proliferation Foxo3a specific (C)). the and three for detect (p-Foxo3a Foxo3a with to proteins and assay p-Foxo3a downstream CCK-8 MEK, related (D) p-MEK, trol. the Akt, of p-Akt, expression (B), the and and LY294002 PI3K/MEK/Erk and and 2HCL PI3K/Akt through proliferation pathways cells U937 signaling regulate might LILRA3 6: Figure vial at available AC etr ltaayi o h niioyecec fGK101,MK-2206- GSK1120212, of efficiency inhibitory the for analysis blot Western (A-C) . * https://authorea.com/users/311535/articles/442241-lilra3-is-increased- p < 0.05, # p < .1vru IR3oeepesn 97clsperae with pretreated cells U937 LILRA3-overexpressing versus 0.01 17 IR3mgtitrc with interact might LILRA3 .