Regulation of CD47 expression on CD14+ monocytes by interferon-α in PBC patients

Background Primary biliary cholangitis (PBC) is a chronic intrahepatic cholestatic autoimmune liver disease characterized by inflammatory injury of small and medium-sized bile ducts in the liver. The pathogenesis of PBC has yet to be entirely understood. CD47/signal-regulatory protein alpha (SIRPα) is closely related to developing autoimmune diseases by promoting inflammatory response. However, the effect of CD47/SIRPα on inflammatory response in PBC patients is still unclear. Objective We investigated the expression of CD47/SIRPα and the effect of inflammatory cytokines on the CD47 expression, analyzed potential autoantibodies against CD47 and the effect of anti-CD47 antibody on the inflammatory response in PBC, provided laboratory basis for the study of the pathogenesis and targets for non-invasive diagnosis and treatment on PBC. Methods The expression levels of CD47 and SIRPα on peripheral blood mononuclear cells (PBMC) were measured in 14 patients with PBC (the PBC group) and 13 healthy subjects (the Control group) by flow cytometry (FCM). The PBMC derived from healthy subjects were stimulated with healthy subjects’ serum, PBC patients’ serum, IFN-α or TNF-α, and the CD47 expression level on CD14+ monocytes was detected by FCM. The level of serum anti-CD47 antibody or IFN-α in PBC patients and healthy subjects was analyzed by ELISA. FCM was used to examine the TNF-α expression level in CD14+ monocytes of healthy subjects stimulated with isotype control antibody, anti-CD47 antibody, LPS or LPS combined with CD47 antibody. Results The CD47 expression level on the CD14+ monocytes in PBC patients was statistically higher than that in the Control group (P<0.01). Compared with the Control group (PBMC+healthy serum), the CD47 expression on CD14+ monocyte stimulated with the PBC patients’ serum (PBMC+PBC patients’ serum) was increased (P<0.001); the CD47 expression on CD14+ monocyte stimulated with IFN-α (PBMC + IFN-α) increased gradually with the increased concentration of IFN-α (P<0.05). However, there was no similar trend on CD14+ monocyte stimulated with the TNF-α (PBMC+TNF-α) (P>0.05). The levels of serum anti-CD47 antibody and IFN-α in the PBC patients were higher than those in healthy subjects (P<0.05). The TNF-α expression level in CD14+ monocyte stimulated with the LPS (PBMC+LPS) or anti-CD47 antibody+LPS group (PBMC+LPS+anti-CD47 antibody) was significantly increased than that in the Control group (PBMC+isotype control antibody) (P<0.01 and P<0.001, respectively). The TNF-α expression level in CD14+ monocyte stimulated with the anti-CD47 antibody + LPS was higher than that with the LPS (P< 0.05). Conclusion The CD47 may be related to the pathogenesis of PBC by inflammatory response. The CD47/SIRPα signal were imbalanced in PBC patients. The presence of serum anti-CD47 antibodies in PBC patients provides a laboratory basis for clinical diagnosis and treatment.


Introduction
Primary biliary cholangitis (PBC) is a chronic intrahepatic cholestatic autoimmune liver disease characterized by inflammatory injury of small and medium-sized bile ducts in the liver.It occurs mainly in middle-aged and older women; the onset is mostly hidden.The early symptoms of the disease are not specific, and it is easy to miss or misdiagnose (1).The pathogenesis of PBC needs to be clarified.The interaction among environmental, genetic, and hormonal factors led to congenital and adaptive immune disorders and loss of self-tolerance (2), leading to antibody and T cell-mediated specific immune attacks against the liver, progressive inflammatory necrosis, and fibrosis of the liver (3,4).The activation of inflammatory mediators such as type I interferon (IFN-I) and tumor necrosis factor (TNF), innate liver lymphocytes, and natural killer T cells played a crucial role in the pathogenesis of the disease (5).In addition, due to the existence of the 'gut-liver axis', when the intestinal mucosa was damaged, Lipopolysaccharides (LPS), the cell wall component of Gram-negative bacteria, entered the liver through the portal vein, which would cause activation of Toll-like receptors 4 (TLR4) and nuclear factor kappa-B (NF-kB) signaling pathway, increased the expression of inflammatory factors, and aggravated the liver from autoimmune damage (6).
CD47 is a transmembrane protein expressed in different cell types, such as thymocytes, T and B cells, monocytes, erythrocytes and nerve cells.It belongs to the immunoglobulin superfamily (7) and is a supramolecular complex composed of integrin, G protein, and cholesterol (8).It interacts with corresponding ligands and mediates cell proliferation, migration, phagocytosis, apoptosis, immune homeostasis, and inhibition of nitric oxide signaling (9,10).signal-regulatory protein alpha (SIRPa) binds to CD47 to initiate an inhibitory signaling pathway, resulting in weakened phagocytosis of macrophages to malignant cells (11).
The binding of CD47 on erythrocyte to the SIRPa on macrophages prevented erythrocyte phagocytosis by suppressing phagocytic activity.The polycythemia phenotype was restored by intercepting CD47-SIRPa through either anti-CD47 treatment or loss of the inhibitory SIRPa-signal in a PV mouse model (12).Both solid and hematologic malignancies expressed higher levels of CD47, which bound with SIRPa to protect the tumor cell against macrophagemediated phagocytosis (13-15).The CD47 enabled cancer cells to escape innate and adaptive immune surveillance leading to metastatic spread, which could be restricted by the administration of anti-CD47 antibodies through affecting tumor growth and tumor microenvironment signaling (16,17).Anti CD47 therapy promoted T cell secretion of pro-inflammatory cytokines in an undifferentiated pleiomorphic sarcoma which expressed highly CD47 (18).
CD47-SIRP a signaling pathway is related to the development of autoimmune diseases.CD47 deficiency improved ocular autoimmune inflammation (19) and furthered macrophagemediated phagocytosis in type I diabetes (20).CD47 facilitated autoimmune valvular carditis through damaging macrophage efferocytosis and increasing cytokine production (21).T cell activation was regulated by macrophages through CD47/SIRPA in inflammatory bowel disease (22).Jin et al. (7) reported that the CD47 expression level on monocytes in SLE patients was significantly increased, promoting SLE patients' inflammatory response.However, the effect of CD47/SIRPa on the inflammatory response in PBC patients has not been reported.
Therefore, the present research clarified the expression of CD47/ SIRPa on mononuclear cells in PBC patients, investigated the effect of serum inflammatory cytokines on the CD47 expression in PBC patients, analyzed potential autoantibodies against CD47 in PBC patients, and explored the role of CD47 and anti-CD47 antibody in the inflammatory response of PBC, provided laboratory basis for the study of the pathogenesis and targets for non-invasive diagnosis and treatment on PBC.

Research object
From December 2021 to February 2023, 14 PBC patients and 13 healthy subjects were selected from Hunan Provincial People's Hospital (the First Affiliated Hospital of Hunan Normal University).Diagnosis of PBC was based on the published American Association for the Study of Liver Diseases (AASLD) criteria, APASL clinical practice guidance: the diagnosis and management of patients with primary biliary cholangitis (2022) (23).Patients were excluded with one or more of the following conditions: (1) severe cardiovascular disease, kidney disease and other serious diseases; (2) acute and chronic infectious diseases and various non-PBC immune system diseases; (3) pregnant or lactating women; (4) liver cancer or other types of malignant tumor.The ethics committee of Hunan Provincial People's Hospital approved the exemption of informed consent, because theresearch used the remaining samples after clinical testing, which did not cause additional harm to patients.

Peripheral blood experiments 2.2.1 Extraction of peripheral blood mononuclear cells
EDTA anticoagulant venous blood of PBC patients or healthy subjects diluted 2-4 times with PBS was mixed with Ficoll lymphatic separation solution at 1:1, centrifuged at 400 g, 18°C-20°C for 40 min.The PBMC layer was taken and centrifuged twice to leave the PBMC for use.

Detection of anti-CD47 antibody level and IFN-a level
According to the instructions, the serum anti-CD47 level and the IFN-a level in the PBC patients and healthy subjects were detected by ELISA kits (#F-111548-A, FANKEW, China) (#F10665-A, FANKEW, China).

Detection of ANA and AMA
Antinuclear antibody (ANA) and antimitochondrial antibody (AMA) were detected by indirect immunofluorescence method [EUROIMMUN Medical Diagnostics (China) Co., Ltd.], autoimmune liver disease antibodies were detected by membrane strip immunoblotting (Suzhou Haobo Co., Ltd.), and serum immunoglobulins by rate scattering turbidimetric assay (Siemens, USA), liver function was detected by the automatic biochemical analyzer (Hitachi Medical Instrument Co., Ltd.).

Statistical analysis
Statistical analysis was performed using Graph Prism 8.3.0 and SPSS Statistics 25 software.Flow data were analysed using the flowjo 6.2 software.The measurement data of normal distribution were expressed as mean ± standard deviation.One-way ANOVA was used to compare the differences among multiple groups, and Post Hoc post hoc test was used for pairwise comparison.The difference between the two groups was analyzed by independent sample t-test or paired t-test.Non-normal distribution data were expressed as median (quartile).Mann-Whitney U test was used for comparison between two groups and multiple groups.P<0.05 indicated a statistically significant difference.

Clinical indicators of patients
A total of 14 patients with PBC and 13 healthy subjects were collected.Among the enrolled 14 PBC patients, 12 cases were positive for ANA, 11 cases were positive for AMA, 1 case was positive for antisoluble liver antigen/hepatopancreatic antigen antibody (SLA/LP), 1 case for anti-smooth muscle antibody (SMA) and 1 case for antihepatocyte cytoplasmic type 1 antibody (LC-1).There was no significant difference in gender and immunoglobulin G between the two groups (P>0.05).The levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin (TBIL), direct bilirubin (DBIL), immunoglobulin M (IgM) and immunoglobulin A (IgA) in PBC patients were higher than those in the control group (P<0.05).In contrast, complement 3 (C3) and complement 4 (C4) were opposite (P<0.05).The clinical indicators of PBC patients and healthy subjects enrolled in this research were shown in Table 1.

The CD47 expression level on CD14 + monocytes incubated with PBC patients serum or recombinant IFN-a
PBMCs of healthy subjects were incubated with healthy subjects serum (n=5), PBC patients serum (n=9), different concentrations of recombinant IFN-a (n=4) or recombinant TNF-a (n=4), and the CD47 expression levels on cultured cells were detected.The CD47 expression level on CD14 + monocytes in the PBC group was significantly higher than that in the Control group (86.06% ± 8.98%, 30.09% ± 16.75%; t=6.94,P=0.001, Figure 5).

3.6
The TNF-a expression level in CD14 + monocytes incubated with the anti-CD47 antibody PBMC in healthy subjects was incubated with an isotype control antibody, anti-CD47 antibody, LPS or anti-CD47 antibody+LPS.The TNF-a expression levels in CD14 + monocytes in cultured PBMC were detected.There were significant differences in the TNFa expression in CD14 + monocytes among the isotype control group, anti-CD47 antibody group, LPS group and anti-CD47 antibody + LPS group (F=76.58,P<0.001).The TNF-a expression level in CD14 + monocytes in the anti-CD47 antibody group was not different from that in the isotype control group (3.76% ± 1.51%, 2.19% ± 0.51%; t=1.70,P>0.05), the TNF-a expression level in CD14 + monocytes in the LPS group was higher than that in the isotype control group (35.43% ± 9.85%, 2.19% ± 0.51%; t=5.84,P<0.01), the TNF-a expression level in CD14 + monocytes in the anti-CD47 antibody + LPS group was higher than that in the LPS group (57.80% ± 3.61%, 35.43% ± 9.85%; t=3.70,P<0.05) (Figure 8).

A B
The CD47 expression levels on CD14 + monocytes incubated with different concentrations of the IFN-a (A) and TNF-a (B).ns: There was no statistical significance between the two groups; ***P< 0.001, **P< 0.01, *P< 0.05.The TNF-a expression levels in CD14 + monocytes in the isotype control group, anti-CD47 antibody group, LPS group and anti-CD47 antibody+LPS group.ns: There was no statistical significance between the two groups; ***P< 0.001, **P< 0.01, *P< 0.05.

Discussion
Immunosurveillance among normal cells, defective cells and foreign pathogens is regulated by cell surface receptors, which mediate the interaction between immune cells and their targets.These ' self ' signal markers interact with proteins expressed on the surface of phagocytes to inhibit phagocytosis (24).CD47 is a protein expressed in almost all body cells, providing a ' do not eat me ' signal to host phagocytes (including neutrophils and macrophages) (25).Cancer cells escaped the recognition and killing of host phagocytes by expressing high levels of CD47 (26,27), indicating that pathogenic results will occur in the case of CD47 overexpression.The present research found increased CD47 expression level on CD14 + monocytes in PBC patients, consistent with the report (7).
When the CD47 and SIRPa are expressed on the monocytes at the same time, the sum of the two signals may determine the final effect of CD47 and SIRPa on cell response (28).Our research showed that the CD47 expression level was increased in PBC patients.In contrast, the expression of SIRPa was normal on CD14 + monocytes in PBC patients, suggesting that immune regulation of the CD47/ SIRPa signals were imbalanced in PBC patients.The research also showed IFN-b and IFN-g/TNF-a decreased erythrophagocytosis by human monocytes in vitro, which was independent from the increase in SIRP-a or SHP-1 expression (29).
Our research also showed that the PBC patients' serum with increased IFN-a level could improve the CD47 expression on CD14 + monocytes from healthy subjects.Recombinant IFN-a also increased the CD47 expression on CD14 + monocytes in a dose-dependent manner.The data showed enhanced expression of IFN-I and toll-like receptor-3 in PBC (30) and IFN-I signaling as a necessary component of the sex bias in murine autoimmune cholangitis (31).The selfderived IFN inducers and a lack of negative feed-back signals downregulating the IFN response contributed to the continuous IFN production in SLE (32).Therefore, we speculated that increased serum IFN-a caused by self-derived IFN inducers or a lack of negative feed-back signals could promote CD47 expression in CD14 + monocytes in PBC patients.
PBC is an autoimmune disease, which can produce a variety of autoantibodies, which helps to diagnose autoimmune diseases.Our research found that the level of anti-CD47 autoantibodies in PBC patients was higher than that in healthy subjects, suggesting that there were autoantibodies against CD47 in PBC patients.Anti-CD47 antibody enhanced the phagocytosis of macrophages by binding to the Fc receptor on the macrophages, which might destroy the interaction of CD47/SIRPa, increase the phagocytic activity of macrophages, lead to an aggravation of experimental autoimmune encephalomyelitis and autoimmune nephritis (33,34).
Studies have shown that the immune response of effector CD4 + T cells (Th1, Th17 and follicular helper T cells) and CD8 + T cells to autoantigens (expressed by hepatocytes and biliary epithelium) was related to PBC pathogenesis (35, 36).Activated Th1 and Th17 cells released various inflammatory factors to promote the occurrence and development of PBC (5).When PBC patients were exposed to LPS due to infection, LPS could bind to cell surface receptors (such as TLR 4/CD14) and induced the secretion of pro-inflammatory cytokines (such as TNF-a, IL-1, IL-6, IL-8), promoting inflammation.Our results showed that the anti-CD47 antibody could not promote TNFa expression in CD14 + monocytes.However, LPS could, and TNF-a expression level was higher when stimulated with the combination of the anti-CD47 antibody and LPS.We assumed that anti-CD47 antibody can enhance the pro-inflammatory effect of LPS, aggravate liver lesions, and form a vicious circle.
In summary, increased inflammatory cytokines promote the expression of CD47 on the CD14 + monocytes in PBC patients.There was anti-CD47 antibody in PBC patients which could enhance the pro-inflammatory effect of LPS, aggravate liver lesions, and form a vicious circle.Blocking the imbalance of CD47/SIRPa signal may contribute to treatment for PBC patients.

TABLE 1
Clinical indicators of the study subject.