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.