A Lectin-EGF antibody promotes regulatory T cells and attenuates nephrotoxic nephritis via DC-SIGN on dendritic cells

Abstract

Background: Interactions between dendritic cells (DCs) and T cells play a critical role in the development of glomerulonephritis, which is a common cause of chronic kidney disease. DC-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN), an immune-regulating molecule of the C-type lectin family, is mainly expressed on DCs and mediates DC adhesion and migration, inflammation, activation of primary T cells. DC-SIGN triggers immune responses and is involved in the immune escape of pathogens and tumours. In addition, ligation of DC-SIGN on DCs actively primes DCs to induce Tregs. Under certain conditions, DC-SIGN signalling may result in inhibition of DC maturation, by promoting regulatory T cell (Treg) function and affecting Th1/Th2 bias.

Methods: A rat model of nephrotoxic nephritis was used to investigate the therapeutic effects of an anti-lectin-epidermal growth factor (EGF) antibody on glomerulonephritis. DCs were induced by human peripheral blood mononuclear cells in vitro. The expression of DC surface antigens were detected using flow cytometry; the levels of cytokines were detected by ELISA and qPCR, respectively; the capability of DCs to stimulate T cell proliferation was examined by mixed lymphocyte reaction; PsL-EGFmAb targeting to DC-SIGN on DCs was identified by immunoprecipitation.

Results: Anti-Lectin-EGF antibody significantly reduced global crescent formation, tubulointerstitial injury and improved renal function impairment through inhibiting DC maturation and modulating Foxp3 expression and the Th1/Th2 cytokine balance in kidney. Binding of anti-Lectin-EGF antibody to DC-SIGN on human DCs inhibited DC maturation, increased IL-10 production from DCs and enhanced CD4+CD25+ Treg functions.

Conclusions: Our results suggest that treatment with anti-Lectin-EGF antibody modulates DCs to suppressive DCs and enhances Treg functions, contributing to the attenuation of renal injury in a rat model of nephrotoxic nephritis.

Figure 1

Effect of PsL-EGFmAb on renal pathology and function. A, PAS staining of renal tissues and corresponding quantification (final magnification × 200). Renal tissues were harvested on day 14. B, Tubulointerstitial damage was evaluated according to the scoring system we mentioned in the Methods. C-F, Twenty-four-hour urine proteins (C), Scr (D) and BUN (E) of nephritic rats were significantly elevated, whereas CCr (F) levels were significantly reduced on day 14 compared with non-nephritic controls (p < 0.01). PsL-EGFmAb treatment significantly reduced twenty-four-hour urine proteins (C), Scr (D), BUN (E) levels and increased CCr (F) compared with NTN group (p < 0.01), indicating improvement of renal function after PsL-EGFmAb treatment. The mean ± SD of three independent experiments is shown. **p < 0.01 vs. control; ^#p < 0.05, ^##p < 0.01 vs. NTN group.

Figure 2

Effect of PsL-EGFmAb on renal DC maturation. A-C, Renal DCs from control (A), NTN (B) and PsL-EGFmAb (C) groups were freshly isolated by MACS using anti-rat OX62 micro-beads on day 14 and maturity of DCs was determined by flow cytometry detected by cell surface expression of MHC class-II, CD80 and DC-SIGN (black line). The isotype-matched controls are shown as gray line. D, Allogeneic mixed lymphocyte reactions were used to evaluate the proliferation of CD4⁺ T cells induced by isolated DCs. MACS-isolated and irradiated DCs from rat kidneys of control, NTN and PsL-EGFmAb-treated groups were co-cultured with CD4⁺ T cells from healthy rats at a 1:10 ratio for five days. T cell proliferation was assessed by [³H]TdR incorporation. The results were shown as fold increases in [³H]TdR incorporation. The mean ± SD of three independent experiments is shown. **p < 0.01 vs. control; ^##p < 0.01 vs. NTN group.

Figure 3

The mRNA expression of cytokines in renal tissues. Real-time PCR for IFN-γ (A), TNF-α (B), IL-6 (C), IL-4 (D), Foxp3 (E), IL-10 (F), TGF-β (G) mRNA expression and IFN-γ/ IL-4 ratio (H) in renal tissues. Expression of the genes of interest was normalized to that of the GAPDH gene, and described as 2^–ΔΔCt. The mean ± SD of four independent experiments is shown. *p < 0.05, **p < 0.01 vs. control; ^#p < 0.05, ^##p < 0.01 vs. NTN group.

Figure 4

PsL-EGFmAb inhibits human DC maturation in vitro. Human CD14⁺ monocytes from healthy donors were isolated using the MACS method and cultured in RPMI 1640 complete medium in the presence of 50 ng/ml human GM-CSF and 20 ng/ml human IL-4 for five days to produce imDCs. mDCs were induced by further treatment with 50 ng/ml TNF-α. In PsL-EGFmAb group, 10 μg/ml PsL-EGFmAb was also added before TNF-α treatment. Expression of HLA-DR, CD80, CD83 and CD86 on imDCs, mDCs, or PsL-EGFmAb-treated DCs was detected by flow cytometry. A, imDCs. B, mDCs. C, PsL-EGFmAb-treated DCs (black line). Appropriate isotype antibodies were used as controls (gray line). Data were representative of at least three independent experiments.

Figure 5

Allogeneic mixed T cell proliferation assays. imDCs, mDCs or PsL-EGFmAb-treated mDCs were co-cultured with freshly isolated human CD4⁺, CD4⁺CD25⁺ and CD4⁺CD25^– T cells, respectively, then proliferation assays were performed and the results are shown as fold increases in [³H]TdR incorporation. Flow cytometric analysis was also performed to detect CD4⁺CD25⁺Foxp3⁺ Tregs. A, DCs co-cultured with CD4⁺ T cells. B, DCs co-cultured with CD4⁺CD25⁺ T cells. C, DCs co-cultured with CD4⁺CD25^– T cells. D, A suppression assay was performed to evaluate the suppressive function of CD4⁺CD25⁺ T cells using the same method. Tregs were either co-cultured with PsL-EGFmAb-treated mDCs (T1) or freshly isolated from health adults. The mean ± SD of four independent experiments is shown. ^#p < 0.05, ^##p < 0.01.

Figure 6

Assays to analyze the cytokines involved in DC maturation. A-E, ELISA assay for IFN-γ (A), IL-12 (B), IL-6 (C), IL-10 (D) and TGF-β (E) using the culture supernatants of imDCs, mDCs (TNF-α-treated DCs) and TNF-α with PsL-EGFmAb-treated DCs in vitro. F, Neutralization assays. PsL-EGFmAb-treated DCs were co-cultured with freshly isolated CD4⁺CD25⁺ Tregs only (control) or in the presence of one of the following neutralizing anti-human antibodies in vitro: anti-IL-6, anti-IL-10, anti-TGF-β, anti-IL-4, anti-TNF-α, anti-IL-12 or anti-IFN-γ mAbs. The isotype-matched controls were also used. Allogeneic mixed T cell proliferation assays were performed as above. The results are shown as fold increases in [³H]TdR incorporation. G, Neutralization assays. CD4⁺CD25⁺ T cells were co-cultured with PsL-EGFmAb-treated mDCs (T1 cell), then mixed with CD4⁺CD25^- effector T cells. Suppression assays were performed in the presence of the neutralizing antibodies mentioned above or with medium only (control). The mean ± SD of three independent experiments is shown. *p < 0.05, **p < 0.01 vs. control; ^#p < 0.05, ^##p < 0.01 vs. mDC group; ⁺p < 0.05, ⁺⁺p < 0.01 vs. imDC group.

Figure 7

PsL-EGFmAb signals to DCs through DC-SIGN. A-C, We generated human monocyte-derived DCs by incubating MACS-isolated CD14⁺ monocytes from healthy donor blood with GM-CSF and IL-4 for 5 days (imDCs). Flow cytometry detected DC-SIGN expression on imDCs using fluorescence-labeled DC-SIGN mAb (A) and PsL-EGFmAb (B) respectively. C, imDCs pre-incubated with anti-DC-SIGN goat antiserum were also included to prevent binding of PsL-EGFmAb to DCs before staining with PsL-EGFmAb. D, Western blotting analysis of DC-SIGN in different samples: imDC lysates immunoprecipitated using PsL-EGFmAb-coated beads (left), imDC lysates without immunoprecipitation (middle), imDC lysates immunoprecipitated using mouse IgG-coated beads (right). Experiments were performed in triplicate and the data from one representative experiment is shown. E, Western blotting analysis of DC-SIGN from different samples: The lysates of imDCs with DC-SIGN siRNA were immunoprecipitated using PsL-EGFmAb-coated beads (left), the lysates of imDCs with DC-SIGN siRNA without immunoprecipitation (middle), the lysates of imDCs with DC-SIGN siRNA immunoprecipitated using mouse IgG-coated beads (right).