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\begin{document}

\title{LILRA3 is increased in IBD patients and functions as an
anti-inflammatory modulator}



\author[1]{Xiucai Lan}%
\author[2]{Fenghua Liu}%
\author[3]{Ying Chang}%
\author[3]{Jing Liu}%
\author[4]{Xiuhua Lan}%
\author[3]{Li Xiang}%
\author[3]{Feng Zhou}%
\author[3]{Qiu Zhao}%
\affil[1]{Zhongshan Hospital Fudan University}%
\affil[2]{Shanghai Xuhui Central Hospital}%
\affil[3]{Wuhan University Zhongnan Hospital}%
\affil[4]{Shanghai Institute of Materia Medica CAS}%


\vspace{-1em}



  
  \date{May 24, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
Growing evidence shows that a homozygous 6.7-kb deletion of the novel
anti-inflammatory molecule leukocyte immunoglobulin like receptor A3
(LILRA3) is associated with many autoimmune disorders. However, its
effects on pathogenesis of inflammatory bowel disease (IBD) have not
been clarified yet. LILRA3 is mainly expressed in monocytes, whereas its
effects on biological behaviors of monocytes have not been
systematically reported. To investigate the association between LILRA3
polymorphism and IBD susceptibility, LILRA3 polymorphism was assessed in
378 IBD patients and 509 healthy controls in our study, quantitative
real-time PCR (qRT-PCR), western blot and immunohistochemistry (IHC)
were employed to detect the LILRA3 expression in IBD patient blood and
intestinal samples. Despite no association of the polymorphism with IBD
development was found, LILRA3 expression was markedly increased in IBD
patients compared with healthy controls. Human U937 monocyte cell line
was employed to establish LILRA3-overexpressing cells and the effects of
LILRA3 on the biological behaviors of U937 cells were systematically
explored. We found that overexpression of LILRA3 in monocytes led to
significant decreases in secretion of interferon-\selectlanguage{greek}γ \selectlanguage{english}(IFN-\selectlanguage{greek}γ\selectlanguage{english}), tumor
necrosis factor-\selectlanguage{greek}α \selectlanguage{english}(TNF-\selectlanguage{greek}α\selectlanguage{english}) and interleukin 6 (IL-6). Additionally, LILRA3
abated monocyte migration by reducing the expression of several
chemokines and enhanced monocyte phagocytosis by increasing CD36
expression. Furthermore, LILRA3 promoted monocyte proliferation through
a combination of Akt and MEK/Erk signaling pathways. We report for the
first time that LILRA3 is related to IBD and functions as an
anti-inflammatory modulator in U937 cells.%
\end{abstract}%



\sloppy


\textbf{Introduction}

Inflammatory bowel disease (IBD), which encompasses ulcerative colitis
(UC) and Crohn's disease (CD), comprises a group of intestinal chronic
disorders characterized by inflammation and periods of remission and
relapse\textsuperscript{{[}1{]}}. The populations of western countries
are more likely to suffer from IBD than those in Asia. However, recent
studies have shown that the incidence and prevalence rates in some Asian
countries are rapidly increasing\textsuperscript{{[}2-4{]}}.

Numerous studies have indicated that genetic factors play important
roles in the pathogenesis of IBD, with involvement of some of these
genes in multiple autoimmune disorders. Leukocyte immunoglobulin like
receptors (\emph{LILRs} ; synonyms: ILT, LIR, CD85) are the most
conserved genes among those located within the leukocyte receptor
cluster on human chromosome 19. The family includes 13 members with
activating or inhibiting capacity\textsuperscript{{[}5{]}}: LILRs with
long cytoplasmic tails bearing tyrosine-based inhibitory motifs are
inhibitory receptors (\emph{LILRBs} ), whereas LILRs with short
cytoplasmic tails have activating functions (\emph{LILRAs}
)\textsuperscript{{[}6,7{]}}. \emph{LILRA3} (ILT-6, CD85e), located in
the centromeric ILT cluster, is a special member of the LILR family.
Genomic sequencing of LILRA3 has revealed that LILRA3 is highly
homologous to other LILRs such as LILRB1 and
LILRB2\textsuperscript{{[}8{]}}, suggesting that LILRA3 might act by
impairing the function of these LILRBs. In addition, LILRA3 shows
presence-absence variation, as opposed to other LILRs, which are
conserved genetically\textsuperscript{{[}9{]}}. For example, some
individuals may carry an aberrant deletion of a 6.7-kb fragment
encompassing the first seven exons\textsuperscript{{[}8,10{]}}, and this
variation has been proven to be associated with many autoimmune
diseases, such as Sj\selectlanguage{ngerman}ögren's syndrome (SS), multiple sclerosis (MS) and
rheumatoid arthritis (RA)\textsuperscript{{[}11-14{]}}.

rs103294 and rs410852 are two single-nucleotide polymorphisms (SNPs) of
the LILRA3 gene. In a case-control study among the Chinese population,
rs103294 was reported to be associated with benign prostatic
hyperplasia\textsuperscript{{[}15{]}}. A genome-wide association study
(GWAS) also identified rs103294 as a new risk locus for prostate
cancer\textsuperscript{{[}16{]}}. This GWAS also revealed that
polymorphism of this locus affects LILRA3 expression. Many recent
articles have demonstrated that the 6.7-kb deletion affects LILRA3 mRNA
and protein expression, with individuals carrying the wild type (+/+)
having much higher levels than those with the homozygous deletion
(-/-)\textsuperscript{{[}17-19{]}}. Nonetheless, increased LILRA3 is
detected in many diseases, such as MS and systemic lupus erythematosus
(SLE)\textsuperscript{{[}18,19{]}}, both of which are autoimmune
disorders characterized by excessive inflammation. These findings
indicate that LILRA3 is a novel susceptibility gene for autoimmune
diseases and might play a crucial role in the pathogenesis of chronic
inflammatory diseases. Additionally, it has been reported that
interleukin 10 (\emph{IL-10} ) or interferon-\selectlanguage{greek}γ \selectlanguage{english}(\selectlanguage{greek}\emph{IFN-γ} \selectlanguage{english}) sharply
upregulates LILRA3 expression in human monocytes, whereas tumor necrosis
factor-\selectlanguage{greek}α \selectlanguage{english}(\selectlanguage{greek}\emph{TNF-α} \selectlanguage{english}) exhibits the opposite
effect\textsuperscript{{[}20{]}}. Furthermore, LILRA3 induces
proliferation of CD8\textsuperscript{+} T-cells and NK cells in the
presence of pro-inflammatory cytokines\textsuperscript{{[}21{]}},
suggesting an anti-inflammatory effect of LILRA3. Apart from
inflammation, LILRA3 is also reported to function as an antagonist of
LILRB2 and to promote synapse formation through the Erk/MEK
pathway\textsuperscript{{[}22{]}}.

Because IBD is an autoimmune disorder characterized by recurrent
intestinal inflammation, we hypothesized that LILRA3 might play a role
in IBD pathogenesis. Accordingly, in this study, we investigated the
interaction between LILRA3 polymorphisms and IBD development. Although
no significant association was found, we surprisingly observed increased
LILRA3 in IBD patients. LILRA3 is mainly expressed in mono-myeloid
cells, such as monocytes, macrophages (M\selectlanguage{ngerman}ø) and dendritic cells
(DCs)\textsuperscript{{[}21,23-25{]}}. Monocytes are critical regulators
in immune responses and have important roles in immune surveillance by
directly phagocytizing pathogens in
circulation\textsuperscript{{[}26{]}}. Monocytes secrete many cytokines
such as IFN-\selectlanguage{greek}γ, \selectlanguage{english}TNF-\selectlanguage{greek}α, \selectlanguage{english}interleukin 6 (\emph{IL-6} ), and IL-10. As these
cells also exert many of their functions outside the vascular system,
crossing the blood vessel wall and migrating to the site of injury are
required\textsuperscript{{[}27{]}}. The effects of LILRA3 on monocytes
have not been systematically reported. We employed the U937 human
monocyte cell line to establish LILRA3-overexpressing cells and then
explored the effects of LILRA3 on the above functions of monocytes as
well as other biological behaviors such as apoptosis and proliferation.

\textbf{Materials and Methods}

\textbf{Ethics Statement}

Our study has been conducted in accordance with the principles expressed
in the Declaration of Helsinki, and was approved by the ethics committee
of Zhongnan Hospital of Wuhan University (2014037). Informed consent has
been obtained.

Patients and samples collection

Lithium sulfate anti-coagulated peripheral blood samples were obtained
from 378 IBD patients (185 CD, 193 UC) and 509 healthy controls. All
patients were recruited from the Department of Gastroenterology,
Zhongnan Hospital of Wuhan University (Wuhan, China) from September 2014
to January 2016. The patients were sub-genotyped according to Montreal
classification criteria. Healthy controls were selected among volunteers
from January to November 2015. The demographic and sub-phenotype data
for the patients and controls are shown in Supplementary Table 1. Total
genomic DNA was extracted from blood using the QIAamp DNA blood midi kit
(Qiagen, Shanghai, Germany). Inflamed ileal and/or colonic tissues were
collected from 36 CD patients and 52 UC patients who underwent endoscopy
at Zhongnan Hospital. All biopsies were taken from inflammatory sites
adjacent to ulcerations. Non-inflamed colonic mucosa samples were
collected from 14 healthy volunteers; another 20 normal biopsies
(Zhongnan Hospital from September 2014 to June 2015) obtained from
uninfected sites 20 centimeters from polyps or neoplasms served as
controls. All patients and controls were of the Han population from
Central China. Diagnosis of CD and UC was based on a combination of
clinical symptoms, laboratory and radiological examinations and
endoscopy with histology.

\textbf{6.7-kb deletion genotyping}

LILRA3 presence-absence variation was detected by polymerase chain
reaction (PCR) using the two sets of primers listed in Supplementary
Table 2. With LILRA3 deletion, primer 1 (flanking the deletion)
amplified a fragment of 166 bp. When LILRA3 was present, primer 2
(designed within the 6.7-kb region) amplified a fragment of 250 bp. Each
DNA sample was tested using the two sets of primers in separate
reactions. The amplified products were analyzed on a 1.5\% agarose gel.
The results of gel electrophoresis are shown in Supplementary Figure 1.

\textbf{rs103294 and rs410852 genotyping}

The genotypes of rs103294 and rs410852 were detected by the PCR-ligation
detection reaction (PCR-LDR) method with technical support from Shanghai
Biowing Applied Biotechnology Company (Shanghai,
China)\textsuperscript{{[}28,29{]}}. The sequences of the primers and
probes are listed in Supplementary Table 2. The PCR product was used as
the template in the LDR reaction, and the LDR products were assessed
using the ABI 3730 sequencer (ABI, Carlsbad, USA).

\textbf{Cell culture}

The human U937 monocyte cell line (Cat. FDCC-HLC063) and 293T cells
(Cat. FDCC-HSN190) were obtained from Fudan Cell Center (F.D.C.C) of
Fudan University (Shanghai, China). Identification of the two cell lines
were at the China Centre for Type Culture Collection in Wuhan, China.
The U937 cells were cultured with RPMI-1640 medium (HyClone, Logan,
USA); 293T cells were cultured in DMEM medium (HyClone, Logan, USA)
containing 10\%fetal bovine serum (FBS) (HyClone, Logan, USA),
penicillin (100 U/ml) and streptomycin (100 \selectlanguage{greek}µ\selectlanguage{english}g/ml) in humidified 5\%
CO\textsubscript{2} at 37\selectlanguage{ngerman}°C.

\textbf{Establishment of the LILRA3-overexpressing U937 cell line}

The open reading frame (ORF) of LILRA3 was amplified from U937 cells
using qRT-PCR and cloned into the expression vector pWSLV-02 (carrying
enhanced green fluorescent protein (EGFP) as a reporter gene)
(Viewsolid, Beijing, China) to generate the plasmid pWSLV-02-LILRA3. For
lentivirus production, 293T cells were co-transfected with the
pWSLV-02-LILRA3 plasmid, which encodes the intact sequence of LILRA3
cDNA, or the empty vector pWSLV-02 as a control using Lenti-Pac HIV
Expression Packaging Kit (GeneCopoeia, Rockville, USA). Virus was
collected at 24 hours and 48 hours after transfection. To prepare the
LILRA3-overexpressing cell line, U937 cells were infected with
lentivirus for 72 hours, and FCM was then used to select cells with
stable LILRA3 lentivirus integration.

\textbf{RNA extraction and quantitative real time polymerase chain
reaction (qRT-PCR)}

Total RNA was extracted from peripheral blood, fresh-frozen biopsies and
pWSLV-02-LILRA3 plasmid- or pWSLV-02-transfected U937 cells using the
Trizol reagent (Invitrogen, Carlsbad, USA). The quantity and quality of
the RNA samples were assessed using a NanoDrop2000 spectrophotometer
(Thermo Scientific, Waltham, USA). Total RNA (1 \selectlanguage{greek}μ\selectlanguage{english}g) was used to
synthesize cDNA using a first-strand cDNA synthesis kit (Thermo
Scientific, Waltham, USA). qRT-PCR was subsequently performed using the
QuantStudioTM6 Flex Real-Time PCR instrument (ABI, Carlsbad, USA) with
the SYBR\selectlanguage{ngerman}\textsuperscript{®} Premix Ex TaqTM II mix (Takara, Kusatsu,
Japan). The 2\selectlanguage{english}\textsuperscript{-ΔΔCT} method was applied to determine the
relative mRNA levels normalized to the housekeeping gene
glyceraldehyde-3-phosphate dehydrogenase (GAPDH).

\textbf{Western blotting}

Total protein was extracted from fresh-frozen intestinal biopsies and
pWSLV-02-LILRA3~plasmid- or~pWSLV-02-transfected U937 cells using a
mixture of RIPA lysis solution containing protease inhibitor and
phosphatase inhibitor. The protein concentration was determined using a
BCA assay kit. RIPA and the BCA kit were purchased from Beyotime
Biotechnology (Shanghai, China). A total of 40 \selectlanguage{greek}µ\selectlanguage{english}g protein from each
sample was separated by 12.5\% or 8\% sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing
conditions and then transferred onto polyvinylidene difluoride (PVDF)
membranes (Millipore, Burlington, USA). The membranes were blocked with
5\% nonfat milk in TBST (Tris-buffered saline (TBS) with 0.1\% Tween 20)
at room temperature for 1 hour and then incubated with specific primary
antibodies in TBST overnight at 4\selectlanguage{ngerman}°C followed by three washes with TBST.
The membranes were then incubated with the following antibodies:
anti-LILRA3 polyclonal antibody (pAb) (Cat. GTX108819, GeneTex, Alton
Pkwy Irvine, USA); anti-CD36 pAb (Cat. 18836-1-AP) and anti-CD206 pAb
(Cat. 18704-1-AP) were obtained from Proteintech (Wuhan, China);
anti-p-Foxo3a mAb (Cat. ab47285) and anti-Foxo3a mAb (Cat. ab17026) were
procured from Abcam (Cambridge, USA); anti-p-Akt monoclonal antibody
(mAb) (Cat. \#4060), anti-Akt mAb (Cat. \#4691), anti-p-MEK1/2 mAb (Cat.
\#9154), anti-MEK1/2 mAb (Cat. \#8727), anti-p-Erk1/2 mAb (Cat. \#4370),
anti-Erk1/2 mAb (Cat. \#4695), anti-p-P38 MAPK mAb (Cat. \#4511),
anti-P38 MAPK mAb (Cat. \#8690), anti-p-PDK1 mAb (Cat. \#3438),
anti-PDK1 pAb (Cat. \#3062), anti-p-c-Raf pAb (Cat. \#9421), anti-p-PI3K
pAb (Cat. \#4228), anti-PI3K mAb (Cat. \#4257), anti-p-P65 mAb (Cat.
\#3033), anti-P65 mAb (Cat. \#8242), anti-p-SAPK/JNK mAb (Cat. \#4668),
anti-JNK2 mAb (Cat. \#9258), anti-p-mTOR mAb (Cat. \#5536), anti-mTOR
mAb (Cat. \#2983) were purchased from Cell Signaling Technology (CST,
Danvers, USA). GAPDH (Cat. KM9002, Sungene Biotechnology, Tianjin,
China) was used as the internal reference. The membranes were then
incubated with a horseradish peroxidase (HRP)-conjugated goat
anti-rabbit/mouse antibody (Cat. LK2001/LK2003, Sungene Biotechnology,
Tianjin, China) in TBST for 2 hours at room temperature. After 3 washes
with TBST, the blots were visualized using enhanced chemiluminescence
kit (Bio-rad, Hercules, USA).

Elisa

Culture media of U937 cells were collected and centrifuged at 20,000 × g
for 1 min. The supernatants were aliquoted and stored at \selectlanguage{english}-80\selectlanguage{ngerman}°C until
analysis. The concentration of IFN-gamma, IL-6, TNF-\selectlanguage{greek}α, \selectlanguage{english}CCL2, CCL3,
CXCL8/IL-8 and CXCL10 was measured using ELISA kits (NeoBiosciences,
Shenzhen, China), according to the manufacturers' protocol.

\textbf{Histopathology, immunohistochemistry (IHC) and
immunofluorescence}

Colon biopsies for histopathology were fixed overnight in 10\% formalin.
After paraffin embedding, 3-\selectlanguage{greek}μ\selectlanguage{english}m sections were cut and stained with
hematoxylin and eosin (H\&E) in accordance with standard protocols for
microscopic examination. IHC was performed on 3 \selectlanguage{greek}μ\selectlanguage{english}m sections of
paraffin-embedded fresh-frozen endoscopic colon tissues from patients
with IBD and control individuals. The sections were deparaffinized, and
antigen was retrieved by microwave oven irradiation using sodium citrate
buffer, followed by blocking with 5\% goat serum for 30 minutes at room
temperature. The sections were incubated with the primary antibody
overnight at 4\selectlanguage{ngerman}°C. After careful washing, the sections were then
incubated for 1 hour at room temperature with a secondary antibody.
Sections treated with phosphate-buffered saline (PBS) instead of the
primary antibody were used as a negative control. Images were acquired
by a microscope (Olympus, Cat. \#IX73). Image-Pro Plus version 6.0
(Media Cybernetics, BetheSEMa, MD) was used to calculate the integrated
option density (IOD).

\textbf{Apoptosis assay}

Flow cytometry (FCM) was applied to detect the involvement of LILRA3 in
apoptosis of U937 cells. U937 cells (4×10\textsuperscript{5}cells/well)
harboring the null vector (pWSLV-02) and overexpression plasmid
(pWSLV-02-LILRA3) were plated in 6-well plates. After 48 hours of
culture, the cells were harvested and washed twice with PBS. The cells
were then stained using an annexin V-PE/7-amino-actinomycin D (7-AAD)
kit (BD, San Jose, USA) following the manufacturer's protocols. Annexin
V-PE was used to detect phosphatidylserine externalization, a hallmark
of early apoptosis, and 7-AAD was used to label DNA fragments, a sign of
cell death. The Q4 area (annexin V-PE positive and 7-AAD negative)
represents early apoptotic cells, and Q2 (both annexin V-PE and 7-AAD
positive) represents late apoptosis.

\textbf{Cell migration}

A migration assay was performed using transwell 24-well Boyden chambers
(Corning, Corning, USA) with an 8.0 \selectlanguage{greek}μ\selectlanguage{english}m pore size membrane without
matrigel. Cells were seeded in the upper chamber at a density of
5\selectlanguage{ngerman}×10\textsuperscript{4}cells/well in 200 \selectlanguage{greek}μ\selectlanguage{english}l serum-free RPMI-1640 medium.
As a chemoattractant, 600 \selectlanguage{greek}μ\selectlanguage{english}l 10\% FBS containing complete medium was
added to the lower chamber. After incubation for 6 hours at 37\selectlanguage{ngerman}°C in
humidified 5\% CO\textsubscript{2}, the upper chamber was detached from
the well, and the cells at the lower chamber, representing migrated
cells, were counted using a cell counting chamber.

\textbf{Phagocytosis assay}

Latex beads (L3030, Sigma, St. Louis, USA) were used to detect the
potential influence of LILRA3 on the phagocytosis capacity of U937
cells. U937 cell lines harboring pWSLV-02-LILRA3 or pWSLV-02 were seeded
in 6-well plates (1.0×10\textsuperscript{5}cells/well) in RPMI-1640
medium containing 1\% FBS, followed by incubation at 37°C in humidified
5\% CO\textsubscript{2} for 20 hours. A mixture of FBS and latex beads
(FBS: latex beads = 20:1) was added to each well. The percentage of
GFP\textsuperscript{+} Texas Red\textsuperscript{+} cells was then
analyzed by FCM at 0.5, 6, 12 and 24 hours.

\textbf{Cell proliferation assay}

Cell proliferation was detected by the cell counting kit-8 (CCK-8) assay
according to the manufacturer's instructions. U937 cells harboring the
LILRA3 overexpression plasmid or the null vector were plated in 96-well
plates (3.0×10\textsuperscript{3}cells/well) for 0, 12, 24, 48 and 72
hours. To each sample, 10 \selectlanguage{greek}μ\selectlanguage{english}l CCK-8 solutions (Dojindo, kamimashiki gun
Kumamoto, Japan) were added at the indicated time. After incubation at
37\selectlanguage{ngerman}°C in humidified 5\% CO\textsubscript{2} for 1 hour, the optical
density (OD) of each sample was measured at 450 nm using an
auto-microplate reader (Infinite M200, Tecan, Manedorf, Switzerland).
Three parallel replicates were employed for each sample.

\textbf{Statistical analysis}

All analyses were performed using SPSS Version 17.0 and GraphPad Prism
software version 5.0. Among the healthy controls, the distributions of
the 6.7-kb deletion genotype, rs103294 and rs410852 were in
Hardy-Weinberg Equilibrium (HWE) (p=0.55, p=0.31, p=0.55 respectively).
The \selectlanguage{greek}χ\selectlanguage{english}2 test was used to analyze the frequencies of alleles and genotypes
between the cases and controls and the differences between the
sub-phenotypes of the patient groups with the controls. Odds ratios
(ORs) are provided with 95\% confidence interval (CI) to estimate the
relative risk of developing CD and UC. Differences in LILRA3 mRNA and
protein expression between the patient groups and controls were analyzed
using one-way analysis of variance (ANOVA). Differences between the U937
cells harboring the null vector and overexpression plasmid were analyzed
using an unpaired t-test. A p value less than 0.05 was considered to be
statistically significant. Data are presented as mean \selectlanguage{ngerman}± SD from three
independent experiments and representative data are shown.

\textbf{Results}

\textbf{The association between LILRA3 gene polymorphism and the
development of IBD}

The deletion rate among 877 subjects enrolled in our study (both
patients and healthy controls) was 89.4\%, which was higher in
comparison with that reported for the Han Chinese population in Beijing
(CHB) (89.4\% \emph{vs.} 70\%)\textsuperscript{{[}30{]}}. The deletion
ratio in three groups was 88.4\% for healthy controls, 91.4\% for CD and
90.7\% for UC, and no association was found between the 6.7-kb deletion
and IBD development (p=0.27, p\textsubscript{FDR}=0.41, OR=1.39,
95\%CI=0.78-2.47 for CD; p=0.39, p\textsubscript{FDR}=0.96, OR=1.28,
95\%CI=0.73-2.22 for UC). In addition, no difference at the allele level
(``-'' \emph{vs.} ``+'') was found (p=0.70, p\textsubscript{FDR}=0.90,
OR=1.05, 95\%CI=0.82-1.35 for CD; p=0.87, p\textsubscript{FDR}=0.87,
OR=1.02, 95\%CI=0.80-1.30 for UC) (Table 1), and the deletion did not
affect the phenotype frequencies of CD or UC (Table 2). These data
indicate that the 6.7-kb deletion has no association with CD and UC
development among the Han population of Central China.

Limited association was detected between the rs103294 and rs410852
genotypes and disease susceptibility. (p=0.94 for rs103294; p=0.17 for
rs410852). Nonetheless, at the allele level, the allele T of rs103294
was found to be a risk locus for CD (p=0.04, p\textsubscript{FDR}=0.12,
OR=1.32, 95\%CI=1.01-1.73) (Table 1). Relationships between the
phenotypes of patient groups and the controls were then analyzed,
revealing that the CD patients carrying the rs410852 genotype were less
likely to develop intestinal stricturing or penetrating complications
(Table 2). However, no association for the genotypes and allele
frequencies between the UC patients and controls was observed for the
groups as a whole or after stratified.

\textbf{IBD patients exhibit increased LILRA3 expression compared with
healthy controls}

The effect of LILRA3 variation on its expression in peripheral blood was
assessed for 36 patients with CD, 48 patients with UC and 53 healthy
controls. The subjects enrolled were derived from samples with available
genotyping data. As shown in Figure 1A, LILRA3 was almost undetectable
in subjects homozygous for the 6.7-kb deletion, whereas its expression
was significantly increased in those heterozygous for the deletion and
with the wild-type gene. In addition, when the homozygous deletion
subjects were excluded, we found remarkable increases in the levels of
LILRA3 mRNA in both the CD (2.11±1.47) and UC (1.72±1.10) patients
compared with the controls (0.98±0.59) (p=0.005 for CD; p=0.014 for UC)
(Figure 1B).

Considering that IBD is a chronic inflammatory disease that primarily
involves the gut, we then carried out qRT-PCR and western blotting to
investigate intestinal LILRA3 expression. Based on our finding that
samples with undetectable LILRA3 expression are homozygous for the
deletion genotype, intestinal samples with undetectable LILRA3 were
directly excluded from this analysis. Overall, a total of 14 healthy
controls, 36 CD and 52 UC samples were included. Increased LILRA3 mRNA
levels were observed in the patient groups compared with healthy
controls. (p \textless{} 0.001 for both CD and UC group) (Figure 1C).
Total protein from another 8 non-IBD, 7 CD and 10 UC samples was
extracted, and in accordance with the qRT-PCR data, western blotting
showed a noteworthy increase in LILRA3 expression in both CD (2.50±1.68)
and UC (2.26±1.72) group in comparison with healthy controls (0.39±0.27)
(p \textless{} 0.05 for both CD and UC group) (Figure 1D).

To further detect pathological changes and in situ expression of LILRA3
in intestinal biopsies, H\&E staining and IHC were applied. The
morphology and structure of mucosa from the healthy controls were
normal. In contrast, the sub-mucosa and lamina propria (LP) of the CD
and UC biopsies were saturated with lymphocytes, plasma cells and
neutrophils (Figure 1E). LILRA3 is reported to exist in a soluble form,
and we consistently found LILRA3 in the cytoplasm of cells located in
the LP (Figure 1E). Compared with the non-IBD controls (0.16±0.009),
many more LILRA3-positive cells were detected in the LP of samples from
the CD (0.21±0.03) and UC patients (0.19±0.03) (p \textless{} 0.01 for
CD; p \textless{} 0.05 for UC) (Figure 1E, 1G). According to previous
study, LILRA3 is mainly expressed in myeloid cells. We then use CD68 and
LILRA3 antibody to stain the macrophages in the LP. Immunofluorescence
assay revealed that the LILRA3 protein were expressed in CD68 positive
macrophages and CD patients possessed more
CD68\textsuperscript{+}LILRA3\textsuperscript{+} cells than healthy
controls (16.09±5.03 for CD, 6.57±1.96 for HC, p \textless{} 0.01)
(Figure 2A-2C). In summary, these findings demonstrate that the
overwhelming majority of LILRA3 protein is expressed in macrophages and
is remarkably increased in IBD patients, identifying a definite role of
LILRA3 in IBD development.

\textbf{Overexpression of LILRA3 downregulates secretion of many
cytokines by U937 cells}

Establishment of LILRA3-overexpressing U937 cells was verified at the
mRNA and protein levels. As shown in Figure 3A, significant increases in
LILRA3 were detected in the overexpressing U937 cells. To explore the
potential effect of LILRA3 on cytokine secretion and further elucidate
its role in inflammation, the supernatant of the two established cell
lines were collected. Elisa assay demonstrated that the presence of
LILRA3 could markedly downregulate secretion of IFN-\selectlanguage{greek}γ \selectlanguage{english}(p \textless{}
0.001), TNF-\selectlanguage{greek}α \selectlanguage{english}(p \textless{} 0.001), and IL-6 (p \textless{} 0.001)
(Figure 3B). In view of that the LILRA3 is a soluble protein, we use
LILRA3-overexpressing U937 cell lines supernate to culture the U937
cells for 12 and 24 hours, and in consistent with the previous results,
we detected significantly decreased in the secretion of IFN-\selectlanguage{greek}γ, \selectlanguage{english}TNF-\selectlanguage{greek}α \selectlanguage{english}as
well as IL-6. Cells cultured for 12 hours showed a more significant
effect of inhibition (Figure 3C). Previous studies have demonstrated an
anti-inflammatory role for LILRA3 based on findings that IL-10
upregulates but TNF-\selectlanguage{greek}α \selectlanguage{english}downregulates LILRA3 production in vitro. Our
results support the growing body of evidence that LILRA3 participates in
inflammation as an anti-inflammatory molecule.

\textbf{The presence of LILRA3 has limited effects on U937 cell
apoptosis}

The impact of LILRA3 on apoptosis has not been reported. After culturing
for 48 hours, cells were harvested and stained with 7-AAD kit, and the
apoptosis ratio was calculated for GFP\textsuperscript{+} cells.
According to the data shown in Figure 3D and 3E, we conclude that
although LILRA3 obviously decreased the late apoptosis rate compared
with the null vector cells (3.64\%\selectlanguage{ngerman}±0.11\% \emph{vs.} 4.41\%±0.20\%, p
\textless{} 0.01), it had little effect on early apoptosis and total
apoptosis frequencies (p \textgreater{} 0.05). This result indicates
that LILRA3 is not involved in regulating apoptosis in U937 cells.

\textbf{LILRA3 attenuates U937 cell migration by decreasing expression
of certain chemokines}

A transwell test was applied to investigate cell migration. We found
that after 6 hours of incubation in 10\% FBS containing RPMI-1640, few
of the U937 cells overexpressing LILRA3 had migrated into the lower
chamber compared with cells harboring the null vector (p \textless{}
0.001) (Figure 3F). Chemokines and their seven-transmembrane, G-protein
coupled receptors are recognized as key mediators physiologically
directing cell migration. We then performed Elisa assay to detect
expression of two CC-type chemokines (CCL2, CCL3) and other two CXC-type
chemokines (CXCL8/IL-8, CXCL10), which were predominantly expressed in
monocytes. Interestingly, we found that CCL2, CCL3, IL-8, CXCL10 were
significantly suppressed by LILRA3 (p \textless{} 0.001) (Figure 3G). To
verify our findings, we then added a certain amount of recombinant CCL2
or CXCL8 (Peprotech, Rocky Hill, USA) to the upper chamber, and the
migration assay results showed that the migration capacity, inhibited by
LILRA3, was reversed by the exogenous IL-8 (p\textless{}0.01) (Figure
3H). The data suggest that LILRA3 attenuates the migration capacity of
U937 cell lines mainly by suppressing IL-8 secretion.

\textbf{LILRA3 promotes phagocytosis of U937 cells by upregulating CD36
expression}

The phagocytosis efficiency was detected by FCM at 0.5, 6, 12 and 24
hours by analyzing the percentage of GFP\textsuperscript{+} Texas
Red\textsuperscript{+} cells. As shown in Figure 4A and 4B,
LILRA3-overexpressing U937 cells showed a strong ability to engulf beads
beginning at 6 hours in comparison with cells expressing the null vector
(5.23\%±0.21\% \emph{vs.} 3.80\%±0.35\%, p \textless{} 0.01 at 6 hours;
26.00\%±0.17\% \emph{vs.} 20.47\%±0.42\%, p \textless{} 0.001 at 12
hours; 30.03\%±0.83\% \emph{vs.} 26.43\%±0.64\%, p \textless{} 0.01 at
24 hours). Pattern recognition receptors (PRRs), mainly including
mannose receptor (MR) and scavenger receptor (SR), play important roles
in phagocytosis. CD206/\emph{MRC1} (an important MR) and
CD36/\emph{SCARB3} (an important class B SR) are two highly effective
endocytic receptors expressed on monocytes\textsuperscript{{[}31,32{]}}.
qRT-PCR and western blotting were employed to further explore whether
LILRA3 could alter expression of these two receptors on the plasma
membrane (for primers used in the reaction, see Supplementary Table 2).
We found that LILRA3 could significantly promote expression of CD36 yet
had no influence on CD206 (Figure 4C, 4D, 4E). CD36 specific antibody
(BD, San Jose, USA) was added to the culture of LILRA3-overexpressing
cell lines, and the phagocytosis efficiency was detected again. The
specific antibody could significantly decrease the phagocytosis ability
of LILRA3-overexpression cell lines at a concentration dependent manner
(p\textless{}0.01 for both groups at 12h culture, p\textless{}0.01 for
1mg/ml and p\textless{}0.001 for 10mg/ml at 24h culture) (Figure 4F).
Taken together, our results reveal that LILRA3 enhances the phagocytosis
ability of U937 cells by increasing CD36 expression.

\textbf{LILRA3 enhances the proliferation of U937 cells through a
combination of Akt and MEK/Erk signaling pathways}

The proliferation capacity of the two established U937 cell lines was
evaluated using CCK-8 assay by detecting OD values at 450 nm at 12, 24,
48 and 72 hours. After 24 hours of culturing, enhanced proliferation was
observed for the LILRA3-overexpressing U937 cells compared with the
cells expressing the null vector, and the difference was quite
significant at 48 and 72 hours (p=0.016 for 48 hours; p=0.003 for 72
hours) (Figure 5A). Western blotting was applied to investigate the
potential signaling pathway, and we found that overexpression of LILRA3
significantly increased the levels of phosphorylated \emph{Akt} ,
\emph{MEK} and \emph{P38 MAPK}(Figure 5B-5D). Given that the MEK and Akt
pathways are closely related to proliferation, our ensuing research
mainly focused on exploring the upstream and downstream factors of these
two kinases. The levels of phosphorylated \emph{Erk} (the accepted
downstream protein of MEK),\emph{PDK1} and \emph{c-Raf} (the classical
kinases for Akt and MEK, respectively), \emph{PI3K} (the conventional
upstream protein of both Akt and MEK), and \emph{Foxo3a} (the shared
downstream protein of MEK and Akt) were further examined. Intriguingly,
we found that phosphorylation of Erk, PDK1, c-Raf and PI3K was
significantly increased but that of Foxo3a decreased in the presence of
LILRA3 (Figure 5C, 5E, 5F and 5G). Phosphorylation of \emph{P65} ,
\emph{SAPK/JNK} and\emph{mTOR} was also investigated, but no difference
was found (see Supplementary Figure 2). Because both Akt and MEK can be
activated by PI3K, we speculated that the two pathways might function
synergistically to regulate U937 cell proliferation. To confirm our
hypothesis, LILRA3-overexpressing cells were seeded into 6-well plates
and pretreated with specific inhibitors of MEK (GSK1120212, 1 \selectlanguage{greek}μ\selectlanguage{english}mol/L),
Akt (MK-2206 2HCL, 5 \selectlanguage{greek}μ\selectlanguage{english}mol/L) or PI3K (LY294002, 20 mmol/L); the three
inhibitors were purchased from Selleckchem (Houston, USA). Western
blotting was used to evaluate the inhibitory efficiency, and the CCK-8
assay was carried out at 24, 48 and 72 hours to detect cell viability.
We found that the three inhibitors could dramatically decrease
phosphorylation of their targeted proteins (Figure 6A-6C) and could
sharply reduce cell viability after 24 hours of culture compared with
cells treated with dimethyl sulfoxide (DMSO) (Figure 6D). Most
importantly, as shown in Figure 6C and 6D, LY294002 (a specific
inhibitor of PI3K) also markedly decreased phosphorylation of Akt and
MEK, with the highest inhibitory efficiency on cell proliferation
compared with the other inhibitors. These results were consistent with
our speculation. Foxo3a is a well-known critical transcription factor
involved in cell growth. Although we detected a noteworthy decline in
phosphorylation of this protein in cells pretreated with the three
inhibitors, this result was not in accordance with our finding when the
level of phosphorylated Foxo3a was detected between
LILRA3-overexpressing and null vector cells. Indeed, Foxo3a
phosphorylation should be enhanced by activated Akt and MEK; in
contrast, we detected a decreased level when LILRA3 was overexpressed. A
possible hypothesis to explain this phenomenon is that LILRA3 might have
a direct/indirect inhibitory effect on Foxo3 phosphorylation; further
investigations are needed to verify this speculation. In conclusion, our
data indicate that LILRA3 regulates cell proliferation, most likely
through PI3K/Akt and PI3K/MEK/Erk signaling pathways.

\textbf{A schematic of the possible signaling pathway for LILRA3}

The exact receptor for LILRA3 remains unknown. The classical human
leukocyte antigen-\selectlanguage{russian}І (HLA-І): \emph{HLA-C} and the non-classical HLA-І
molecule \emph{HLA-G} as well as \emph{Nogo 66} (a highly conserved
66-amino acid loop of \emph{Nogo A} ) are reported as possible receptors
for LILRA3\textsuperscript{{[}22,33,34{]}}. Based on these findings, we
hypothesize that LILRA3 might activate the above signaling pathway by
interacting with one of the above or unknown receptors (Figure 7).

\textbf{Discussion}

Our initial goal was to clarify the association between LILRA3
polymorphism and IBD susceptibility. Although no significant association
with IBD development was observed between rs103294, rs410852 and the
6.7-kb deletion genotypes, we demonstrated that homozygous 6.7-kb
deletion results in undetectable LILRA3 levels, which is consistent with
previous studies\textsuperscript{{[}18,19{]}}. In addition, we detected
a much higher deletion ratio among Han population of Central China
compared with CHB. Unexpectedly, we found LILRA3 expression in IBD
patients was significantly higher than in healthy controls. LILRB1 and
LILRB2 are two members of LILRB family. LILRB1 was reported to block the
combination of CD8 molecular with HLA-I hence to regulate
CD8\textsuperscript{+} T cells. LILRB2 promoted hematopoietic stem cell
(HSC) proliferation. Considering that LILRA3 is homologous to LILRB1 and
LILRB2, additionally, LILRA3 alleles were in strong linkage
disequilibrium with LILRB2 alleles. Previous study reported that LILRA3
might affect functions of LILRB2 and LILRB1. Based on these findings, we
speculate that LILRA3 might play certain roles in immune system.

LILRA3, a member of the highly homologous family of receptors primarily
co-expressed on mono-myeloid leukocytes, is increasingly recognized as
regulating innate immune responses\textsuperscript{{[}24,35{]}}. In
vitro, recombinant LILRA3 shows strong and specific binding to the
surface of primary peripheral blood mononuclear cells (PBMCs), U937
monocyte cells and B cells\textsuperscript{{[}23{]}}. Additionally,
recombinant LILRA3 could dramatically abrogate lipopolysaccharide
(LPS)-induced TNF-\selectlanguage{greek}α \selectlanguage{english}secretion by monocytes, and increases in LILRA3 have
been detected in MS and SLE patients. All these data indicate a critical
role for LILRA3 in inflammatory diseases. The intestinal LP contains a
diverse array of mononuclear phagocyte (MNP) subsets, including
conventional dendritic cells, monocytes and tissue-resident macrophages
that collectively play an essential role in mucosal homeostasis,
infection and inflammation. In our study, we also found that LILRA3 is
mainly expressed on macrophages located in the LP of intestinal and CD
patients possessed more
CD68\textsuperscript{+}LILRA3\textsuperscript{+}cells. Intestinal
macrophages are mainly derived from monocytes in circulation, and LILRA3
was reported to mainly expressed on monocytes. Since IBD is
characterized by immunologic disorder and monocytes are critical
regulators in immune responses and played important roles in IBD
pathogenesis. We speculate that LILRA3 might exert certain function on
monocytes. To deduce the effect of LILRA3 on immune responses, in our
study, human U937 monocytes were employed to establish
LILRA3-overexpressing cells, and we observed that LILRA3 could markedly
decrease IFN-\selectlanguage{greek}γ, \selectlanguage{english}TNF-\selectlanguage{greek}α \selectlanguage{english}and IL-6 secretion and increase IL-10 secretion.
This finding further confirms an anti-inflammatory effect of LILRA3 in
immune responses.

Monocytes account for 2--10\% of all leukocytes in human body. These
cells circulate through the blood and lymphatic system and are recruited
to damaged tissue or infected sites to differentiate into M\selectlanguage{ngerman}ø and DCs,
thus triggering adaptive immune responses\textsuperscript{{[}36{]}}.
Therefore, stimulation of monocyte migration can help to boost
inflammatory responses. In vivo, migration is largely mediated by
interaction between chemokines and chemokine receptors. We found that
the migration ability of U937 cells was sharply attenuated by LILRA3.
CCL2, CCL3, CXCL8 and CXCL10 are the main chemokines secreted by
monocytes, and LILRA3 was able to conspicuously reduce expression of
these molecules on U937 cells. This finding is in agreement with our
transwell assay, which demonstrated impaired migration capacity of U937
cells when LILRA3 was overexpressed. Based on these results, we
speculate that LILRA3 might prevent monocyte migration from the
circulation into tissues, thereby suppressing inflammation.

In this study, LILRA3-overexpressing cells engulfed more latex beads
than cells harboring the null vector. This result indicates that LILRA3
can promote the phagocytosis capacity of U937 cells. Phagocytosis is
predominantly mediated by endocytic receptors present on the phagocyte
membrane\textsuperscript{{[}37{]}}. SR and MR are important receptors
associated with endocytosis. SR consists of a family of receptors with
many ligands and can be divided into two subgroups: class A and class B.
Class A and class B can be further divided into many types including
SR-AI, SR-AII, SR-AIII, SR-BI, SR-BII, and
SRBIII\textsuperscript{{[}29,37{]}}. SRBIII, also termed CD36, is mainly
expressed on cells of lymphoid and hematopoietic lineages, such as
monocytes, Mø, platelets, endothelial cells and a variety of cultured
cell lines\textsuperscript{{[}38,39{]}}. MR is a carbohydrate-binding
receptor mainly expressed by Mø and DCs\textsuperscript{{[}32{]}}. In
our study, both CD36 and CD206 were detected on U937 cells, and LILRA3
increased CD36 expression but had a limited effect on CD206 expression.
These results are consistent with our phagocytosis assay.

Similar to previous studies reporting that recombinant LILRA3 could
induce the proliferation of CD8\textsuperscript{+} T-cells and NK cells
in mixed lymphocyte reactions (MLR)\textsuperscript{{[}21{]}}, we
observed that increased LILRA3 expression could enhance U937 cells
proliferation in vitro. PI3K/Akt and PI3K/MEK/Erk signaling are two
essential pathways involved in cell survival and
growth\textsuperscript{{[}40,41{]}}, and we found that LILRA3 activated
these two pathways to regulate cell proliferation. Foxo3a, a
tumor-suppressive transcriptional factor, is known to regulate various
cellular events such as metabolism, proliferation, tumorigenesis, cell
cycle arrest, apoptosis and longevity\textsuperscript{{[}42-44{]}}.
Previous studies have shown that the two key kinases Akt and Erk have
the ability to regulate the transcriptional activity of Foxo3a via
specific phosphorylation\textsuperscript{{[}45-47{]}}. Akt- or
MEK-mediated phosphorylation causes Foxo3a to bind to the 14-3-3
protein, followed by translocation from the nucleus to the cytoplasm,
with inhibition of transcriptional
activity\textsuperscript{{[}43,44,48{]}}. The Ras/Erk pathway also
regulates cell growth by inhibiting Foxo3a via MDM2-mediated
degradation\textsuperscript{{[}44{]}}. In our study, we observed that
Foxo3a phosphorylation was decreased when LILRA3 was overexpressed. In
contrast, phosphorylation of Foxo3a was conspicuously reduced when
LILRA3-overexpressing cells were pre-incubated with specific inhibitors
of MEK, Akt and PI3K. One possible explanation for this inconsistency is
that LILRA3 might directly/indirectly affect Foxo3a phosphorylation
through an unknown mechanism. Further experiments are needed to
elucidate this mechanism.

Taken together, we found that LILRA3 might function as an
anti-inflammatory molecule in U937 cells based on the following: 1)
LILRA3 directly inhibited inflammation by decreasing proinflammatory
cytokine secretion; 2) LILRA3 restrained monocytes in circulation system
by attenuating their migration ability and accordingly reduced Mø and
DCs in tissues, thereby restricting activation of adaptive immunity; 3)
increased LILRA3 enabled monocytic cells to engulf more pathogens in
circulation; 4) LILRA3 increased the proliferation of immunocytes, such
as monocytes, CD8\textsuperscript{+} T-cells and NK cells, thus allowing
these cells to kill more pathogens and microorganisms.

Collectively, for the first time, we report that LILRA3 expression is
markedly increased in IBD patients. LILRA3 might function as an
anti-inflammatory modulator in innate immune responses and regulate cell
proliferation through a combination of Akt and MEK/Erk signaling
pathways. Further efforts are needed to explore the exact role of LILRA3
in adaptive immunity.

\textbf{Acknowledgments}

This work was supported by grants from National Natural Science
Foundation of China (grant number 81470823, 81800488).

The authors thank all individuals who participate in this study.

\textbf{Conflicts of interest statement}

There is no financial conflict of interest to declare for any of the
authors.

\emph{}

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39. Greenwalt, D.E., R.H. Lipsky, C.F. Ockenhouse, H. Ikeda, and G.A.
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40. Furukawa-Hibi, Y., Y. Kobayashi, C. Chen, and N. Motoyama,\emph{FOXO
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Factor.} Cell, 1999. 96(6): p. 857-868.

\textbf{Table 1: Association between LILRA3 polymorphism and IBD
development}\selectlanguage{english}
\begin{longtable}[]{@{}lllllllllllllll@{}}
\toprule
\begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
Name\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
\textsuperscript{a}HC (N=509)\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
CD (N=185)\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
CD (N=185)\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
CD (N=185)\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
CD (N=185)\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
UC (N=193)\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
UC (N=193)\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
UC (N=193)\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
UC (N=193)\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
UC (N=193)\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
UC (N=193)\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
UC (N=193)\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
N (\%)\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
N (\%)\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
\emph{P}\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
\emph{\textsuperscript{b}P}\textsubscript{FDR}\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
OR (95\%CI)\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
N (\%)\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
N (\%)\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
\emph{P}\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
\emph{P}\textsubscript{FDR}\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
\emph{P}\textsubscript{FDR}\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
OR (95\%CI)\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
OR (95\%CI)\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
\textbf{6.7 kb deletion} -/-,-/+\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
450(88.4)\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
169(91.4)\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
175(90.7)\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
+/+\textsuperscript{c}*\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
59(11.6)\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
16(8.6)\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
0.27\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
0.41\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
1.39(0.78-2.47)\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
18(9.3)\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
18(9.3)\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
0.39\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
0.96\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
0.96\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
1.28(0.73-2.22)\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
1.28(0.73-2.22)\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
Allele\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
-\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
660(64.8)\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
244(65.9)\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
252(65.3)\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
252(65.3)\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
+* \textbf{rs103294} TT, CT CC* Allele T C* \textbf{rs410852} GG, AG AA*
Allele G A*\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
358(35.2) 453(89.0) 56(11.0) 696(68.4) 322(31.6) 495(97.2) 14(2.8)
864(84.9) 154(15.1)\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
126(34.1) 165(89.2) 20(10.8) 274(74.1) 96(25.9) 176(95.1) 9(4.9)
315(85.1) 55(14.9)\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
0.70 0.94 \textbf{0.04} 0.17 0.90\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
0.90 0.94 0.12 0.40 0.90\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
1.05(0.82-1.35) 1.02(0.59-1.75) \textbf{1.32(1.01-1.73)} 0.55(0.24-1.30)
1.02(0.73-1.43)\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
134(34.7) 172(89.1) 21(10.9) 257(66.6) 129(33.4) 187(96.9) 6(3.1)
336(87.0) 50(13.0)\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
134(34.7) 172(89.1) 21(10.9) 257(66.6) 129(33.4) 187(96.9) 6(3.1)
336(87.0) 50(13.0)\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
0.87 0.96 0.52 0.80 0.30\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
0.87 0.96 0.78 0.96 0.78\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
0.87 0.96 0.78 0.96 0.78\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
1.02(0.80-1.30) 1.01(0.60-1.72) 0.92(0.72-1.18) 0.88(0.33-2.33)
1.20(0.85-1.69)\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
1.02(0.80-1.30) 1.01(0.60-1.72) 0.92(0.72-1.18) 0.88(0.33-2.33)
1.20(0.85-1.69)\strut
\end{minipage}\tabularnewline
\bottomrule
\end{longtable}

\textsuperscript{a}HC: healthy controls

\emph{\textsuperscript{b}P} \textsubscript{FDR}: represents the
\emph{P}value adjusted by false discovery rate correction.

\textsuperscript{c}*: represents the wild genotype or wild allele.

\textbf{Table 2: Association between LILRA3 polymorphism and IBD in
subphenotype-control cohort}\selectlanguage{english}
\begin{longtable}[]{@{}llllllllllllllllllllll@{}}
\toprule
\begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
Phenotype\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
6.7 kb deletion\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
6.7 kb deletion\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
6.7 kb deletion\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
6.7 kb deletion\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
rs103294\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
rs103294\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
rs103294\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
rs103294\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
rs103294\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
rs103294\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
rs410852\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
rs410852\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
rs410852\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
rs410852\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
rs410852\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
rs410852\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
rs410852\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
rs410852\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
-/-,-/+ \emph{VS} +/+\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
-/-,-/+ \emph{VS} +/+\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
\emph{P}\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
OR(95\%CI)\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
TT,CT \emph{VS} CC\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
TT,CT \emph{VS} CC\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
\emph{P}\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
\emph{P}\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
OR(95\%CI)\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
OR(95\%CI)\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
GG,AG \emph{VS} AA\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
GG,AG \emph{VS} AA\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
GG,AG \emph{VS} AA\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
\emph{P}\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
\emph{P}\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
OR(95\%CI)\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
OR(95\%CI)\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
\textsuperscript{d}\textbf{HC (N)}\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
450\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
59\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
Ref\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
Ref\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
453\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
56\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
56\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
Ref\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
Ref\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
Ref\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
Ref\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
495\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
14\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
14\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
Ref\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
Ref\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
Ref\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
\textbf{CD (N)}\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
169\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
16\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
165\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
20\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
20\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
176\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
9\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
9\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
\emph{Diagnosis age \textsuperscript{e}(yr)} \textless{}17\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
13\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
2\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
1.00\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
0.79(0.19-3.87)\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
12\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
2\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
2\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
1.00\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
1.00\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
0.74(0.16-3.40)\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
0.74(0.16-3.40)\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
14\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
0\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
0\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
1.00\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
1.00\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
1.03(1.01-1.04)\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
17-40\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
122\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
10\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
1.60(0.80-3.22)\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
113\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
13\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
13\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
0.83\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
0.83\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
1.08(0.57-2.03)\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
1.08(0.57-2.03)\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
120\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
6\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
6\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
0.38\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
0.38\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
0.57(0.21-1.50)\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
\textgreater{}40\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
34\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
4\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
1.00\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
1.11(0.38-3.25)\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
40\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
5\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
5\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
1.00\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
1.00\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
0.99(0.38-2.61)\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
0.99(0.38-2.61)\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
42\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
3\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
3\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
0.31\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
0.31\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
0.40(0.11-1.43)\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
\emph{Site of disease}\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
L1 Ileal L2 Colonic L3 Ileocolitis L4 Upper \textsuperscript{f}GI\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
45 51 69 4\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
3 8 5 0\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
0.26 0.66 0.21 1.00\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
1.97(0.59-6.53) 0.84(0.38-1.85) 1.81(0.70-4.67) 1.13(1.10-1.17)\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
43 51 67 4\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
5 8 7 0\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
5 8 7 0\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
0.90 0.56 0.69 1.00\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
0.90 0.56 0.69 1.00\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
1.06(0.40-2.80) 0.79(0.36-1.75) 1.18(0.52-2.70) 1.12(1.09-1.16)\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
1.06(0.40-2.80) 0.79(0.36-1.75) 1.18(0.52-2.70) 1.12(1.09-1.16)\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
45 56 71 4\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
3 3 3 0\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
3 3 3 0\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
0.36 0.55 0.80 1.00\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
0.36 0.55 0.80 1.00\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
0.42(0.12-1.53) 0.53(0.15-1.89) 0.67(0.19-2.39) 1.03(1.01-1.04)\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
\emph{Disease behavior}\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
B1 Non-stricturing/ Non-penetrating B2 Stricturing B3 Penetrating
Perianal disease \textbf{UC (N)}\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
114 33 22 26 175\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
11 4 1 1 18\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
0.37 1.00 0.46 0.34\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
1.36(0.69-2.67) 1.08(0.37-3.16) 2.88(0.38-21.79) 3.41(0.45-25.59)\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
113 31 21 25 172\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
12 6 2 2 21\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
12 6 2 2 21\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
0.65 0.49 0.99 0.79\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
0.65 0.49 0.99 0.79\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
1.16(0.60-2.25) 0.64(0.26-1.60) 1.30(0.30-5.68) 1.55(0.36-6.70)\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
1.16(0.60-2.25) 0.64(0.26-1.60) 1.30(0.30-5.68) 1.55(0.36-6.70)\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
122 33 21 26 187\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
3 4 3 1 6\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
3 4 3 1 6\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
1.00 \textbf{0.03} \textbf{0.04} 0.54\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
1.00 \textbf{0.03} \textbf{0.04} 0.54\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
1.12(0.33-4.07) \textbf{0.23(0.07-0.75)} \textbf{0.20(0.05-0.74)}
0.74(0.09-5.81)\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
\emph{Diagnosis age (yr)} \textless{}17 17-40 \textgreater{}40
\emph{Disease location} E1 Proctitis E2 Left-sided E3 Extensive\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
3 96 76 19 70 86\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
0 8 10 4 8 6\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
1.00 0.25 0.99 0.61 0.73 0.15\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
1.13(1.10-1.17) 1.57(0.73-3.40) 1.00(0.49-2.03) 0.62(0.21-1.89)
1.15(0.53-2.50) 1.88(0.79-4.49)\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
22 73 77 19 68 85\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
0 10 11 4 10 7\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
0 10 11 4 10 7\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
0.20 0.78 0.68 0.54 0.64 0.33\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
0.20 0.78 0.68 0.54 0.64 0.33\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
1.12 (1.09-1.16) 0.90 (0.44-1.85) 0.87 (0.43-1.73) 0.59 (0.19-1.79) 0.84
(0.41-1.73) 1.50 (0.62-3.41)\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
1.12 (1.09-1.16) 0.90 (0.44-1.85) 0.87 (0.43-1.73) 0.59 (0.19-1.79) 0.84
(0.41-1.73) 1.50 (0.62-3.41)\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
2 101 84 22 76 89\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
0 2 4 1 2 3\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
0 2 4 1 2 3\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
1.00 0.90 0.57 0.49 1.00 1.00\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
1.00 0.90 0.57 0.49 1.00 1.00\strut
\end{minipage} & \begin{minipage}[t]{0.02\columnwidth}\raggedright\strut
1.03 (1.01-1.04) 1.43 (0.32-6.38) 0.59 (0.19-1.85) 0.62 (0.08-4.95) 1.08
(0.24-4.82) 0.84 (0.24-2.98)\strut
\end{minipage}\tabularnewline
\bottomrule
\end{longtable}

\textsuperscript{d}HC: healthy controls

\textsuperscript{e}yr: year

\textsuperscript{f}GI: gastrointestinal

\textbf{Figure Legends}

\textbf{Figure 1: LILRA3 is increased in IBD patients compared with
healthy controls.} (A) Impact of the 6.7-kb deletion on LILRA3 mRNA
expression in peripheral blood. mRNA expression was evaluated by qRT-PCR
assay. Data are expressed as the mean \selectlanguage{ngerman}± SD. Sample groups and genotypes
were indicated. \textsuperscript{**} p \textless{}
0.01,\textsuperscript{***} p \textless{} 0.001 versus same genotypes in
healthy controls (HC). (B) mRNA expression of LILRA3 in blood when
samples homozygous for the deletion (-/-) were excluded. (C) mRNA
expression of LILRA3 in the intestine. Subjects possessing undetectable
LILRA3 levels were excluded. (D) Protein expression of LILRA3 in the
intestine. Protein was extracted from 8 non-inflammatory bowel disease
(NIBD), 7 CD and 10 UC samples. GAPDH was used as a loading control. (E)
H\&E staining (the left two columns) and immunohistochemistry for LILRA3
(the right column) were performed on 3 \selectlanguage{greek}μ\selectlanguage{english}m colon sections, and
representative pictures are shown. Scale bar is 50 \selectlanguage{greek}μ\selectlanguage{english}m for the left
column, 30 \selectlanguage{greek}μ\selectlanguage{english}m for the right two columns. (F) Negative control staining
for immunohistochemistry. Sections were treated with PBS instead of the
primary antibody. Scale bar is 30 \selectlanguage{greek}μ\selectlanguage{english}m. (G) Quantification of
LILRA3\textsuperscript{+} cells in intestinal biopsies. A total of 20
NIBD, 14 CD and 14 UC samples were enrolled. Each experiment was
repeated for at least three times. *p \textless{}
0.05,\textsuperscript{**}p \textless{} 0.01, \textsuperscript{***}p
\textless{} 0.001 versus HC or NIBD groups.

\textbf{Figure 2: LILRA3 is mainly expressed on
CD68\textsuperscript{+}macrophages in human intestinal.} (A)
Localization of LILRA3 expression by Immunofluorescence. CD68 monoclonal
antibody was used to mark macrophages. LILRA3 protein were expressed in
CD68\textsuperscript{+}macrophages and CD patients possessed more
CD68\textsuperscript{+}LILRA3\textsuperscript{+} cells than Non-IBD
group. Scale bar is 20 \selectlanguage{greek}μ\selectlanguage{english}m for each column. (B) Negative control staining
for immunofluorescence. Sections were treated with PBS instead of the
primary antibody. (C) Quantification of
LILRA3\textsuperscript{+}CD68\textsuperscript{+} cells in intestinal
biopsies. Each group includes 5 samples. Each experiment was repeated
for at least three times. \textsuperscript{*}p \textless{} 0.05,
\textsuperscript{**}p \textless{} 0.01, \textsuperscript{***}p
\textless{} 0.001.

\textbf{Figure 3: Effect of LILRA3 on U937 cells and related
mechanisms.} (A) Verification of LILRA3 expression in the two
established cells by qRT-PCR, Elisa and western blotting. (B) Effect of
LILRA3 on cytokine secretion by U937 cells. Expression of IFN-\selectlanguage{greek}γ, \selectlanguage{english}TNF-\selectlanguage{greek}α
\selectlanguage{english}and IL-6 was assessed by Elisa. Data are expressed as mean \selectlanguage{ngerman}± SD. (C)
Supernate collected from the LILRA3-overexpressing U937 cell lines was
used to culture the U937 cells, and secretion of IFN-\selectlanguage{greek}γ, \selectlanguage{english}TNF-\selectlanguage{greek}α \selectlanguage{english}as well as
IL-6 were significantly decreased. Cells cultured for 12 hours showed a
more significant effect of inhibition. (D) Representative flow cytometry
analysis of apoptosis stained with annexin V-PE and 7-AAD among
LILRA3-overexpressing U937 cells and U937 cells expressing the null
vector. (E) Statistical analysis of apoptotic rate (\%) in the two
established cell lines. (F) Effect of LILRA3 on U937 cell migration.
Cell counts migrated into the lower chamber were analyzed between the
two cell lines. (G) Impact of LILRA3 on chemokine secretion in U937
cells by Elisa assay. CCL2, CCL3, IL-8 and CXCL10 were dramatically
decreased by LILRA3. (H) Recombinant CCL2 and CXCL8 were added to the
upper chamber in the migration assay, and LILRA3 induced impaired cell
migration was reversed by the exogenous IL-8. Data are expressed as mean
\selectlanguage{ngerman}± SD. Each experiment was repeated for at least three
times.\textsuperscript{*}p \textless{} 0.05, \textsuperscript{**}p
\textless{} 0.01, \textsuperscript{***}p \textless{} 0.001.

\textbf{Figure 4: LILRA3 enhances the phagocytosis ability of U937 cells
by upregulating CD36 plasma membrane expression} . (A) Representative
FCM analysis of phagocytosis by U937 cells at 0.5, 6, 12 and 24 hours.
The percentage of GFP\textsuperscript{+} Texas Red\textsuperscript{+}
cells was analyzed. (B) Phagocytosis rates were statistically analyzed
between LILRA3-overexpressing U937 cells and U937 cells expressing the
null vector. (C, D and E) Effects of LILRA3 on CD36 and CD206 expression
according to qRT-PCR and western blotting. (F) The specific antibody of
CD36 could significantly decrease the phagocytosis ability of
LILRA3-overexpression cell lines at a concentration dependent manner.
Each experiment was repeated for at least three
times.\textsuperscript{*}p \textless{} 0.05, \textsuperscript{**}p
\textless{} 0.01, \textsuperscript{***}p \textless{} 0.001.

\textbf{Figure 5: LILRA3 increases U937 cell proliferation and the
possible signaling pathway} . (A) Proliferation of the
LILRA3-overexpressing U937 cells and null vector U937 cells were
analyzed by CCK-8 assay. OD at 450 nm was detected at 0, 12, 24, 48 and
72 hours. (B, C and D) Western blot analysis of p-Akt, Akt, p-MEK, MEK,
p-Erk, Erk, p-P38 MAPK and P38 MAPK in LILRA3 overexpressing cells (OE)
and null vector cells (NV). (E, F and G) Western blot analysis of
p-PDK1, PDK1, p-c-Raf, p-PI3K, PI3K, p-Foxo3a and Foxo3a, p-Foxo4,
p-Foxo1 in LILRA3 overexpressing cells (OE) and null vector cells (NV).
Each experiment was repeated for at least three
times.\textsuperscript{*}p \textless{} 0.05, \textsuperscript{**}p
\textless{} 0.01, \textsuperscript{***}p \textless{} 0.001 versus null
vector cells. GAPDH abundance was used as a control.

\textbf{Figure 6: LILRA3 might regulate U937 cells proliferation through
PI3K/Akt and PI3K/MEK/Erk signaling pathways} . (A-C) Western blot
analysis for the inhibitory efficiency of GSK1120212, MK-2206-2HCL and
LY294002 and the expression of the related downstream proteins (p-Foxo3a
and Foxo3a for (A) and (B), p-Akt, Akt, p-MEK, MEK, p-Foxo3a and Foxo3a
for (C)). GAPDH abundance was used as a control. (D) CCK-8 assay to
detect the proliferation of LILRA3-overexpressing U937 cells (OE) after
pretreated with three specific inhibitors for indicated time. NV: Null
vector U937 cells. Each experiment was repeated for at least three
times. \textsuperscript{*}p \textless{} 0.05, \textsuperscript{\#}p
\textless{} 0.01 versus LILRA3-overexpressing U937 cells pretreated with
DMSO.

\textbf{Figure 7: A schematic of the possible signaling pathway of
LILRA3} . LILRA3 might interact with one of the listed or unknown
receptors and trigger PI3K/Akt and PI3K/MEK/Erk signaling to regulate
U937 cell proliferation. LILRA3 might directly/indirectly inhibit Foxo3a
phosphorylation. Whether Foxo3a plays a role in LILRA3-induced cell
proliferation needs to be further investigated.

\textbf{Hosted file}

\verb`Figures.doc`  available at \url{https://authorea.com/users/311535/articles/442241-lilra3-is-increased-in-ibd-patients-and-functions-as-an-anti-inflammatory-modulator}

\selectlanguage{english}
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\end{document}

