Background
Systemic lupus erythematosus (SLE) is an autoimmune disease in which altered T cell function and polyclonal B cell activation followed by autoantibody production including anti-double stranded DNA antibodies (anti-dsDNA), thus, leading to immune complex deposition in multiple organs, particularly in the kidney. These depositions drive local inflammatory responses that can lead to tissue damage and clinical disease [
1,
2].
Granulocyte colony-stimulating factor (G-CSF) is a growth factor for neutrophils, that can accelerate neutrophil reconstitution after bone marrow suppression and activate effector functions of mature neutrophils [
3]. G-CSF also stimulates proliferation, differentiation, and peripheral mobilization of hematopoietic stem cells [
3]. Furthermore, G-CSF has been reported to modulate both T cell and innate immune responses. G-CSF can induce Tr1 cells in vitro, and mobilize CD4
+CD25
+Foxp3 regulatory T cells (Tregs) [
4,
5]. In addition, G-CSF can mobilize human tolerogenic dendritic cells and induce human semi-mature dendritic cells, and thereby induce type 1 regulatory T (Tr1) cells through interleukin (IL)-10 release [
6,
7]. G-CSF suppresses production of IL-1β, IL-12, interferon (IFN)-γ, and tumor necrosis factor (TNF)-α, but increases serum levels of IFN-α and IL-10 [
7,
8]. Recently, G-CSF was also reported to induce CD11b
+Gr-1
+ myeloid-derived suppressor cells (MDSC), a subset of innate-like suppressor cells [
9,
10]. MDSCs are a heterogeneous population of immature myeloid cells that can suppress both T cells and natural killer (NK) cells and thereby suppress autoimmunity as well as tumor immunity [
10,
11]. MDSCs are divided into two subsets, granulocytic MDSCs (CD11b
+Ly6G
+Ly6C
low), and monocytic MDCSs (CD11b
+Ly6G
-Ly6C
high) [
10]. However, CD11b
+Ly6G
+Ly6C
low and CD11b
+Ly6G
-Ly6C
high cells could be inflammatory granulocytes and inflammatory monocytes, respectively, because these populations share surface markers with MDSCs.
Despite the immunomodulatory effects of G-CSF, results of G-CSF treatment in SLE are still controversial. Low-dose G-CSF treatment accelerated lupus nephritis in MRL lymphoproliferation (MRL/lpr) strain mice and increased autoantibody production in B6.S
le1.Sle2.Sle3 spontaneous mouse model of lupus [
12,
13]. In contrast, high-dose G-CSF treatment prevented lupus nephritis and delayed mortality [
13]. In patients, G-CSF induced disease flares in both lupus nephritis and cutaneous lupus [
14,
15]. These controversial results require further study to confirm the role of G-CSF in lupus nephritis and to determine the involved mechanisms. In this study, we investigated whether G-CSF can ameliorate lupus nephritis in a NZB/W F1 mouse lupus model, and examined the related mechanisms.
Methods
Animals and treatment regimens
NZB/W F1 mice spontaneously develop a disease closely resembling human SLE [
16]. Female NZB/W F1 mice were purchased from SLC Inc. (Hamamatsu, Japan) and housed under the pathogen-free conditions. The experimental group was injected subcutaneously with recombinant human G-CSF (Grasin, Kyowa Kirin, Korea) for 5 consecutive days every week from 24 weeks of age, at a dose of 250 μg/kg/day during 12 weeks. In low-dose experiments, 250 μg/kg/day of human G-CSF was administered 3 times a week for 12 weeks from 24 weeks of age. The control group received only phosphate-buffered saline (PBS) injection. For Treg depletion, depleting anti-CD25 antibodies (PC61, Bio X Cell, West Lebanon, NH, USA) were administered at a dose of 0.5 mg 3 times a week from 33 to 36 weeks.
Measurement of proteinuria, renal function, anti-dsDNA, complement component 3 (C3), and cytokines
Spot urine proteinuria was measured using protein reagent strips (URiSCAN; Yongdong Pharmaceutical Co., Seoul, Korea) once per week for 12 weeks. The measurement was semi-quantitative: 0 = none or trace amount of proteinuria, 1+, 30–100 mg/dL; 2+, 100–300 mg/dL; 3+, 300–1000 mg/dL; 4+, ≥ 1000 mg/dL. Urine albumin concentrations were measured using a mouse albumin ELISA kit (Alpco Diagnostics, Salem, NH, USA) and normalized to urine creatinine concentrations. Serum levels of BUN and creatinine were measured at 32 and 36 weeks using QuantiChrom urea and creatinine assay kits (BioAssay Systems, Hayward, CA, USA) [
17]. Serum concentration of mouse anti-dsDNA and C3 were measured using ELISA kits (Alpha Diagnostic International, San Antonio, TX, USA; Abcam, Cambridge, MA, USA) at 36 weeks. Levels of monocyte chemoattractant protein-1 (MCP-1) and cytokines (IL-6, TNF-α, IL-2, IFN-γ, IL-10, IL-4 and IL-17) were measured at 36 weeks in both serum and renal tissues using cytometric bead array kits (BD Biosciences, San Diego, CA, USA).
Renal histologic analysis
Paraffin sections of fixed kidneys were stained with Periodic Acid-Schiff’s (PAS) stain kit, and evaluated according to described protocol [
18,
19]. Briefly, glomerular pathology was evaluated by assessing 20 glomerular cross-sections (gcs) per kidney, and each glomerulus was scored on a semiquantitative scale: 0 = normal (35–40 cells/glomerular cross-sections, gcs); 1 = mild (glomeruli with few lesions, slight proliferative changes, mild hypercellularity, 41–50 cells/gcs); 2 = moderate (glomeruli with moderate hypercellularity, 50–60 cells/gcs, segmental and/or diffuse proliferative changes, hyalinosis); 3 = severe (glomeruli with segmental or global sclerosis, and/or exhibiting severe hypercellularity (>60 cells/gcs), necrosis, crescent formation). Interstitial/tubular pathology was assessed semiquantitatively on a scale of 0–3 in 10 randomly selected high-power fields. We determined the largest and average number of infiltrates and damaged tubules and subsequently adjusted the grading system accordingly: 0, normal; 1, mild; 2, moderate; 3, severe. Perivascular cellular accumulation was determined semiquantitatively by scoring the number of cell layers surrounding the majority of vessel walls (0, none; 1, < 5; 2, 5–10; 3, ≥ 10 cell layers).
Kidney cryostat sections were stained with goat anti-mouse IgG (Sigma-Aldrich) or rabbit anti-mouse C3 (Abcam) for 4 h. Then, they were incubated at room temperature with Alexa Fluor 488 donkey anti-goat IgG or Alexa Fluor 568 donkey anti-rabbit IgG (Molecular Probe; Invitrogen USA) for 1 h. Deposition of IgG and C3 within the peripheral glomerular capillary walls and the mesangium was measured as the mean fluorescence in 10 glomeruli per mouse. Scores were assigned based on the intensity of IgG/C3 deposition (0–3+), where 0 represents no deposition and 3 denotes intense deposition [
20]. All histologic analysis was performed by two independent pathologists blinded to the treatment group.
Flow cytometry
Renal leukocytes and splenocytes were pretreated with anti-mouse CD16/32 (clone 2.4G2) to block nonspecific Fc binding. Kidney leukocytes were identified by labeling cells with CD45 (30-F11). The fluorochrome-conjugated antibodies (BD Biosciences or eBioscience) were used for flow cytometric analysis: CD45 (30-F11), CD3 (145-2C11), CD4 (RM4-5), CD8 (53-6.7), CD25 (ebioPC61), CD44 (IM7), CD69 (H1.2 F3), Foxp3 (ebio), CD11b (M1/70), Gr-1 (RB6-BC5, ebio), Ly6G (RB6-8c5), Ly6C (AL-21), CD19 (eBio1D3), CD138 (281-2), CXCR5 (SPRCL5), PD-1 (J43), NK1.1 (PK136). Appropriate fluorochrome-conjugated isotype-matched irrelevant monoclonal antibodies were used as negative controls. 7-aminoactinomycin D was also added to distinguish between live and dead cells. The stained cells were analyzed using BD FACSCanto II (BD Biosciences).
Statistical analysis
Data were expressed as the mean ± standard error of the mean (SEM). Student’s t-test was used to compare means. Survival rates were analyzed using the Kaplan-Meier method and compared by the log-rank test. Difference in proteinuria during treatment was analyzed using generalized estimating equations. P values of less than 0.05 were considered statistically significant. All data were analyzed using SPSS v.22.0 software (SPSS Inc., Chicago, IL, USA).
Discussion
This study demonstrated that G-CSF treatment decreased proteinuria, serum levels of BUN and creatinine, and ultimately decreased mortality of NZB/W F1 mice through the expansion of Tregs. G-CSF treatment also decreased serum levels of anti-dsDNA, increased serum levels of C3, and attenuated renal tissue injury including deposition of IgG and C3 in NZB/W F1 mice. Furthermore, G-CSF treatment induced expansion of CD4+CD25+Foxp3+ Tregs, with decreased renal infiltration of T cells, B cells, inflammatory granulocytes and monocytes rather than MDSCs. G-CSF treatment also decreased expression of MCP-1, IL-6, TNF-α, IL-2, IL-10, and IL-17.
Chronic treatment of low-dose G-CSF (10 μg/kg) aggravated lupus nephritis in MRL/
lpr mice, where these results might be attributed to increased Th2 cytokines and IFN-α [
3,
7,
13]. Conversely, chronic treatment of high-dose G-CSF (200 μg/kg) attenuated lupus nephritis in MRL/
lpr mice by potentially decreasing glomerular expression of FcRγIII and IL-12 production [
13]. The G-CSF dose in this study was comparable to the high-dose of G-CSF in the previous study [
13]. When we reduced G-CSF dose by 40 %, we could not ameliorate lupus nephritis. Proinflammatory chemokines (MCP-1) and cytokines (IL-6, TNF-α), Th1 cytokines (IL-2, IFN-γ), Th2 cytokines (IL-4, IL-10), and Th17 cytokines (IL-17A) are involved in pathogenesis of lupus nephritis, and are therapeutic targets for lupus nephritis [
23,
24]. G-CSF treatment decreased the levels of these pathogenic chemokine/cytokines such as MCP-1, IL-6, TNF-α, IL-2, IL-10, and IL-17, and thus were consistent with its suppressive effects on both innate and adaptive immune cells in NZB/W F1 mice. Despite reduced IL-10 levels, reduced levels of IL-6 in both serum and renal tissues might have contributed towards Treg expansion by G-CSF treatment. Taken together, these results indicated that the suppression of various proinflammatory cytokines involved in lupus nephritis pathogenesis could have led to the G-CSF-mediated attenuation of lupus nephritis.
G-CSF decreased inflammatory granulocytes (CD11b
+Ly6G
+Ly6C
low) or monocytes (CD11b
+Ly6G
-Ly6C
high) in both kidney and spleen. These results were consistent with previous studies in murine lupus models. In those studies, the percentage of CD11b
+Gr-1
low monocytes in the kidney increased as the disease progressed in the MRL/lpr mouse model of lupus, and it expanded in B6 mice with established chronic graft-versus-host disease [
12,
25]. Although inflammatory granulocytes and monocytes share surface markers with the MDSCs, these data suggested that G-CSF treatment decreased inflammatory granulocytes and monocytes rather than MDSCs in parallel with improvement of lupus nephritis.
We found that Tregs expanded in the G-CSF group and Treg depletion abrogated the G-CSF-mediated beneficial effects on proteinuria in NZB/W F1 mice. These findings were consistent with a previous study, which demonstrated that G-CSF recruited Tregs, and thereby prevented onset of spontaneous diabetes in NOD mice [
26]. CD4
+CD25
+ Treg cells were decreased in SLE patients and Treg cell numbers were inversely correlated with disease activity [
27,
28]. Moreover, infusion of Tregs attenuated disease progression and reduced mortality in murine lupus [
29]. Therefore, we assumed that Treg expansion could be the beneficial mechanism of G-CSF on lupus nephritis in this study. Although MDSC could contribute to induction of Tregs [
30], lack of MSDC expansion might exclude this mechanism in our setting. It was also observed that G-CSF decreased CD25
-Foxp3
+ T cells in contrast to CD25
+Foxp3
+ Tregs. These results demonstrated another benefit of G-CSF treatment for lupus nephritis, because CD25
-Foxp3
+ T cells were increased in the case of active lupus nephritis and had lower suppressive activity than CD25
+Foxp3
+ Tregs [
31,
32].
Beneficial effects of G-CSF for lupus nephritis observed in this study suggest G-CSF as potential treatment for lupus nephritis, considering G-CSF is already used in clinical treatment of neutropenia after chemotherapy. Human G-CSF is less potent in mice than murine G-CSF, thus we used a very high dose of human G-CSF in NZB/W F1 mice. We expect to reduce this dose to tolerable levels in clinical trials.
Most patients with lupus nephritis take long-term immunosuppressants such as corticosteroids, cyclophosphamide, or mycophenolate mofetil. Therefore, one of the limitations of this study is that we did not evaluate the interacting effects of G-CSF with standard immuosuppressants for lupus nephritis. Further studies are needed to confirm our findings in clinically plausible situations.
Acknowledgement
We would like to thank Kyowa Kirin for the generous supply of G-CSF.