Elevated Serum Interleukin-34 Level in Patients with Systemic Lupus Erythematosus Is Associated with Disease Activity

We measured the interleukin-34 (IL-34) level in sera from patients with systemic lupus erythematosus (SLE) and discoid lupus erythematosus (DLE) using an enzyme-linked immunosorbent assay (ELISA). Blood tests, including assays to determine C-reactive protein (CRP), complement (C) 3, C4, immunoglobulin (Ig) A, IgG, IgM, anti-double-stranded DNA antibody (Anti-dsDNA Ab) and hemoglobin (Hb) levels and white blood cell (WBC) and platelet (PLT) counts, were performed using standard methods. Lupus nephritis (LN) was diagnosed according to the American College of Rheumatology (ACR) renal criteria. The SLE disease activity was scored using the SLE Disease Activity Index (SLEDAI). Among the 110 SLE cases, IL-34 could be detected in 79 cases (71.8%). IL-34 was barely detected in the control group. The serum level of IL-34 was significantly higher in the SLE group. No change was observed in the serum IL-34 concentration in the SLE patients regardless of LN status. Correlations were observed between the serum IL-34 level and the disease activity parameters. The SLE patients with detectable IL-34 levels had higher SLEDAI and IgG concentrations and lower C3 and Hb levels than patients with undetectable IL-34 levels. Therefore, IL-34 could be a potential disease activity marker for SLE.

Serum IL-34 level and disease activity in SLE patients. A significant positive correlation was observed between the IL-34 levels and the disease activity marker SLEDAI (r = 0.319, p = 0.004) ( Fig. 2A) but not with CRP. In contrast, a statistically significant negative correlation was observed between IL-34 and C3 in the SLE patients (r = 0.324, p = 0.004) ( Fig. 2A). Thus, the IL-34 level could be correlated with the SLE disease activity. Furthermore, the serum IL-34 level was correlated with IgG and anti-dsDNA antibody production in SLE patients (r = 0.259, p = 0.021; r = 0.352. p = 0.001, respectively) (Fig. 2B). Thus, IL-34 might be associated with antibody production in SLE pathogenesis.
Because hematological changes are quite common in SLE patients, the IL-34 level is significantly negatively associated with Hb and PLT (r = 0.334, p = 0.003; r = 0.236, p = 0.038, respectively) (Fig. 2C). No correlation was observed between the IL-34 level and the WBC count.
No difference was found in PLT between the two groups.

Discussion
This study is the first to show that the IL-34 level is elevated in SLE patients and correlated with the disease activity. In this study, we reported that the serum IL-34 level was significantly elevated in the SLE patients compared to that in the healthy controls. Among the SLE patients with or without LN involvement, no statistically significant difference was observed in the IL-34 level. However, according to Bethunaickan R, the expression of IL-34 is increased in mice with LN 14 , and IL-34 might play a role in the pathogenesis of LN. The current data are not sufficient to reach such a conclusion, and more evidence is required. Moreover, in our study, the IL-34 level is significantly positively correlated with the disease activity as assessed by the SLEDAI. The serum C3 level decreases in SLE patients, particularly in patients with an active disease status. In this study, the serum IL-34 level was correlated with the decreased C3 level. Furthermore, the levels of SLEDAI in the IL-34 + group were much higher than those in the IL-34group. In contrast, the C3 level in the IL-34 + group was much lower. Altogether, the serum IL-34 level might be associated with the disease activity in SLE.
Furthermore, we showed correlations among IL-34, IgG and anti-dsDNA Ab. Thus, IL-34 might be associated with antibody production in SLE pathogenesis.
Because SLE is a highly heterogeneous disease, characterizing subgroups of SLE patients with specific disease phenotypes could help clarify the pathogenesis of SLE. Cytokine measurements might lead to the characterization of these pathways and the identification of potential therapeutic targets. Hematological involvement is quite common in patients with SLE. In this study, the IL-34 level was negatively correlated with hemoglobin and exhibited a weak negative association with platelets. However, in the subsequent analysis of the groups with the IL-34+ & IL-34− SLE patients, a difference in hemoglobin, but not in platelets, was observed. Thus, IL-34 may be associated with anemia in SLE patients. However, more studies are needed to clarify whether IL-34 plays a role in the hematological changes observed in SLE patients.
Elevated concentrations of inflammatory mediators are characteristic of autoimmune diseases with chronic or recurrent inflammation. IL-34 plays pivotal roles in the proliferation and differentiation of mononuclear phagocyte lineage cells and osteoclastogenesis and is necessary for the maintenance of Langerhans cells after the resolution of inflammation 14,15 . Recently, IL-34 has been shown to play important roles in the pathogenesis of RA 8,9 . IL-34 is overexpressed in the inflamed salivary glands of patients with Sjogren's syndrome (SS) 16 . Furthermore, IL-34 may play a role in cancer. IL-34 promotes tumor progression and metastatic processes in osteosarcoma via the induction of angiogenesis 17 . However, our study has limitation for not including a disease control group, such as another non-SLE autoimmune disease. Therefore, elevated levels of IL-34 may be non-specific features of systemic inflammation and not a specific pathogenic factor of lupus. Further studies on IL-34 and other inflammation diseases are needed.
Both SLE and RA are systemic autoimmune diseases. Although functional studies are clearly required to confirm the role of IL-34 in the pathogenesis of SLE, the observed correlations between the elevated levels of IL-34 and SLEDAI, antibody production and anemia suggest that IL-34 plays a role in the modulation of immune inflammatory pathways in SLE.
Many cytokines play important roles in SLE 18 . TNF-α and IL-6 are major inflammatory mediators of pathology in both SLE and RA 18,19 . TNF-α also plays a role in anemia. Furthermore, IL-6 10,11 , IP10 12 and MCP-1 13 participate in the pathogenesis of SLE. IL-34 can induce the expression of IL-6, IP10 and MCP-1 in human whole  7 . The expression of IL-34 increases with inflammation in patients with inflammatory bowel disease and experimental colitis and is associated with an elevated TNFα and IL-6 expression 20 . Altogether, we hypothesize that IL-34 might play a role in the pathogenesis of SLE by promoting the production of other pro-inflammatory cytokines, including TNF-α, IL-6, IP10 and MCP-1.
In summary, the serum IL-34 level was significantly elevated in the SLE patients and correlated with the disease activity and homological changes. IL-34 could be a potential disease activity marker, and this study might have revealed new insight for the study of SLE disease activity.

Methods
Patients. This study was approved by the ethics committee of the 1 st Affiliated Hospital of China Medical University. The experiments were conducted in accordance with the regional Ethics Committee guidelines and regulations. Informed consent forms were obtained from all patients.
Serum was obtained from 110 patients with SLE and 31 patients with discoid lupus erythematosus (DLE), which is the most common chronic cutaneous lupus subtype. The diagnosis of SLE was based on the American College of Rheumatology criteria for SLE 21 In addition, 55 healthy age-and gender-matched controls were recruited. All serum samples were stored at −70 °C until analysis. Since drugs can affect the circulating IL-34 levels, the serum was collected only from SLE patients who were not under treatment. Among the 110 SLE patients, eighty-nine had newly diagnosed SLE, and the other twenty-one were previously diagnosed but had not taken corticosteroids or immunosuppressive drugs for at least three months before enrollment in the study. All thirty-one DLE patients were newly diagnosed, and no drugs were used. Blood tests, including assays to determine C-reactive protein (CRP), complement (C) 3, C4, immunoglobulin (Ig) A, IgG, IgM, anti-double-stranded DNA antibody (Anti-dsDNA Ab) and hemoglobin (Hb) levels and white blood cell (WBC) and platelet (PLT) counts, were performed using standard methods. LN was identified if there was evidence of involvement according to the ACR renal criteria. The disease activity was scored using the SLE Disease Activity Index (SLEDAI) 22 . Statistical analysis. All data are presented as the mean ± SD (parametric) or median (range) (non-parametric). The t-test and the non-parametric Mann-Whitney test were performed to compare the variables between groups. Pearson's or Spearman's correlation coefficient was used to test the correlations. A multivariable linear regression analysis was also performed to assess the independent predictive value of IL-34 in disease activity and hematological changes in the SLE patients. All analyses were performed using SPSS 17.0 and GraphPad 6 software.