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 < 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 < 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 < 0.01 for CD; p < 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+LILRA3+ cells
than healthy controls (16.09±5.03 for CD, 6.57±1.96 for HC, p
< 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.