Impact of CD200-Fc on dendritic cells in lupus-prone NZB/WF1 mice

Abnormal expression of CD200/CD200R1 may contribute to the immunologic abnormalities in patients with systemic lupus erythematosus (SLE). This study aimed to assess the function of CD200/CD200R1and impact of CD200-Fc on dendritic cells in lupus-prone NZB/WF1 mice. Female NZB/WF1 mice were treated with CD200-Fc or control for 4 weeks. Plasma samples were collected to measure autoantibody levels. The expression levels of CD200/CD200R1 in peripheral blood mononuclear cells (PBMCs) and splenocytes were examined. The percentage of CD200/CD200R1-positive cells in splenocytes from NZB/WF1 mice was lower than that of C57BL/6 mice (p < 0.05). The plasma level of anti-dsDNA was significantly higher in NZB/WF1 mice than C57BL/6 mice (p < 0.001). However, the anti-dsDNA levels decreased (p = 0.047) after CD200-Fc treatment. Finally, CD200-Fc reduced the levels of IL-6 (p = 0.017) and IL-10 (p = 0.03) in the dendritic cell culture supernatant. This study suggests that the immunosuppressive CD200/CD200R1 signaling pathway might be involved in the immunopathology of NZB/WF1 mice; the present results merit further exploration of agents that can modulate the CD200/CD200FR1 pathway as a therapy for human lupus.

peripheral blood mononuclear cells (PBMCs) and subtypes of DCs and to treat lupus-prone NZB/WF1 mice with intraperitoneal injections of recombinant CD200-Fc proteins to investigate the effects of intervening with the CD200 pathway in SLE.

Materials and Methods
Mice and treatment. Female C57BL/6 and NZB/WF1 mice were purchased from Weitonglihua (Beijing, China) and the Jackson Laboratory (Bar Harbor, ME, USA), respectively, and were used at 20 to 22 weeks of age. All mice were housed under specific pathogen-free (SPF) conditions. All procedures were performed according to the National Institutes of Health Guide for Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee at the Peking Union Medical College Hospital, Chinese Academy of Medical Sciences.
All mice were divided into the following three groups: 1) NZB/WF1 group: five female NZB/WF1 mice were intraperitoneally injected with normal saline (1 mL/mouse); 2) CD200-Fc group: five NZB/WF1 mice were intraperitoneally injected with CD200-Fc recombinant protein (10 μ g/mouse); 3) five age-and sex-matched C57BL/6 mice were defined as the controls and were intraperitoneally injected with normal saline (1 mL/mouse). All mice were treated for 4 weeks at 2-day intervals, and samples of spleen and peripheral blood were collected after treatment.
Flow cytometric analysis (FCM). Venous blood was collected from the orbital sinus of each mouse after anesthesia, and PBMCs were prepared by Ficoll-Hypaque density-gradient centrifugation. Cell-surface antigen expression on the splenocytes and PBMCs were determined using mAbs purchased from eBioscience (San Diego, CA, USA) unless otherwise stated; the mAbs included CD200 (OX90.1), CD11c (N418), CD11b (M1/70),CD45R (B220) (RA3-6B2), and CD200R1 (OX 110) (R&D system, (Minneapolis, MN,USA)and were either unlabeled or labeled with phycoerythrin, fluorescein isothiocyanate, or allophycocyanin. In all experiments, negative isotype control mAbs of the respective IgGs were included to allow accurate background limits, and all incubations were performed and maintained at 4 °C.
Enzyme-linked immunosorbent assay (ELISA). The ELISA assay was performed to detect the level of anti-nuclear antibody (ANA), anti-dsDNA and anti-RNP/Sm antibodies (Alpha Diagnostic International, San Antonio, TX, USA) in murine plasma diluted at 1:100. The supernatants of cultured Pan DCs were pooled from replicate wells and subjected to ELISA assays to detect the production of cytokines, including IL-6, IL-10, IL-12 and TNF-α (eBioscience, San Diego, CA, USA). All detections were performed according to the manufacturer's instructions.
Histologic assessment of kidneys and urinary protein assays. The kidneys of the mice were harvested, fixed in 10% neutral-buffered formalin, paraffin embedded and cut into 3.5-μ m-thick sections. All sections were stained with Periodic Acid-Schiff (PAS). The glomerular pathology was scored based on the previous study as follows: 0, normal (35-40 cells/gcs); 1, few lesions with slight proliferative changes and mild hypercellularity (41-50 cells/gcs); 2, moderate hypercellularity (51-60 cells/gcs), segmental and/or diffuse proliferative changes, hyalinosis, and moderate exudates; and 3, severe hypercellularity (> 60 cells/gcs) with segmental or global sclerosis and/or severe necrosis, crescent formation, and heavy exudation 17 . Finally, urinary protein was determined using Multistix (Bayer-Siemens, Erlangen, Germany) and a urine analyzer. Statistical analysis. All data were analyzed using SPSS 21.0 software (SPSS Inc., Chicago, IL, USA). An independent-sample t test was used to compare the differences between the groups or differences before and after treatment. P-values < 0.05 were considered statistically significant.

Discussion
It is well recognized that the current therapy for chronic autoimmune diseases, especially SLE, is relatively nonspecific and has limited efficacy. Our previous study demonstrated that CD200 and CD200R1 expression and function are abnormal in SLE and may contribute to the immunologic abnormalities in SLE 16 . In the present study, we conducted new observations by selecting NZB/WF1 mice and examining the therapeutic potential of the recombinant CD200-Fc protein in lupus-prone mice. Our study demonstrated that the percentage of CD200 + cells in splenocytes and PBMCs of NZB/WF1 mice was significantly lower than that of C57BL/6 mice. CD200-Fc treatment successfully reduced the levels of anti-dsDNA in the plasma, decreased cytokine production, including IL-6 and IL-10, by DCs and ameliorated renal histopathology. This suggests that CD200 and CD200R1 may play a role in the prevention of SLE and that CD200-Fc is a potential effective therapeutic agent. On the other hand, recent studies revealed that tolerogenic DCs (tolDCs) are special DCs with a promising immunotherapeutic potential for restoring self-tolerance in autoimmune diseases especially SLE 18,19 . However, the effects of CD200-Fc on tolDCs remain to be clarified.
Lupus-prone NZB/WF1 mice, which were generated as F1 hybrids between the NZB and NZW strains, have long been used as a classical model of systemic lupus erythematosus. NZB/WF1 mice are characterized by increased levels of autoantibodies, including anti-dsDNA IgG, and glomerulonephritis that becomes apparent at  20 . Autoantibodies, especially the most sensitive autoantibodies to dsDNA, play a central role in the onset and development of SLE. The detection of anti-dsDNA autoantibodies improved our ability to diagnose SLE and active disease. Overproduction of autoantibodies and the subsequent hyperactivity of the immune response is believed to be caused by insufficient clearance of apoptotic material by macrophages and DCs 3 . Our findings demonstrated that the level of anti-dsDNA decreased significantly in the CD200-Fc-treatment group, which indicated a role of CD200-Fc in ameliorating the disease activity of SLE.

5-6 months of age
Lupus nephritis (LN) is one of the most significant causes of morbidity and mortality among patients with SLE, and it is the major cause of death of NZB/WF1 mice 21 . The findings in this study demonstrated that NZB/ WF1 mice exhibited glomerular hypercellularity, proliferative glomerulonephritis, higher PAS scores and a higher level of urine protein compared with C57BL/6 mice. The results demonstrated that NZB/WF1 mice developed active LN. For the CD200-Fc group, the histopathology of proliferative glomerulonephritis was ameliorated, with lower PAS scores compared with the NZB/WF1 group. In the study, the difference in urine protein among the NZB/WF1 and CD200-Fc groups was unremarkable, but there appeared to be a decreasing trend after CD200-Fc treatment.
Elevated levels of IL-6 and IL-10 have been associated with modification of the endogenous levels of autoantibodies, including anti-dsDNA, and these increased levels have been shown to be crucial for the development of SLE, whereas blockage of the IL-6 and IL-10 receptors inhibits or delays the onset of renal damage in NZB/WF1 mice 3,22-24 . An in vitro study demonstrated that incubation of lymphocytes with CD200-Fc inhibits the lymphocyte reaction and alters cytokine production, with decreased production of IL-2 and IFN-gamma and increased  production of IL-4 andIL-10 25 . In this study, our findings demonstrated that CD200-Fc reduces the levels of IL-6 and IL-10 produced by DCs. These results may imply that CD200-Fc has a different impact on the production of cytokines, especially IL-10, in different types of cells.
Inappropriate TLR signaling in DCs has been confirmed to be associated with autoimmunity. Up-regulation of TLR4 can disrupt immunological tolerance, which plays a major role in innate immunity and, consequently, induces a lupus-like autoimmune disease in a mouse model and in patients with SLE 26,27 . Our study also demonstrated that TLR4 was significantly higher in the DCs of lupus-prone NZB/WF1 mice compared with C57BL/6 mice. However, there was no remarkable difference in the TLR4 expression in the CD200-Fc group compared with the NZB/WF1 group.
In conclusion, we speculate that CD200/CD200R1 might play a general immunosuppressive role and that CD200-Fc may be of value for the treatment of autoimmune disorders caused by dysfunction of DCs in NZB/ WF1 mice. These findings are helpful for addressing the increasing interest in exploring the immunotherapeutic feasibility of targeting the CD200/CD200R1 signaling pathway for the management of SLE. To our knowledge, this is the first study to investigate the therapeutic potential of CD200-Fc in SLE, and the findings require further preclinical and clinical exploration. Compared with C57BL/6, the levels of IL-6 (A), IL-10 (B), (IL-12) and TNF-α (D) were higher in the plasma of NZB/WF1 mice, while the levels of IL-6 (A) and IL-10 (B) in the CD200-Fc group were lower than in the NZB/ WF1 group. The means and interquartile ranges are depicted as solid lines.