Lipocalin-2 Exacerbates Lupus Nephritis by Promoting Th1 Cell Differentiation

Abstract

Background: Lipocalin-2 (LCN2) is an indicator of the severity of lupus nephritis (LN) and plays a pivotal role in immune responses, but it is not known if its effect on LN pathogenesis derives from regulating the immune imbalance of T lymphocyte subsets.

Methods: The expression of LCN2 in T cells and kidneys was assessed in renal biopsies from patients with LN. We investigated the relationship between LCN2 levels and development of LN and systemic illness by injecting anti-LCN2 antibodies into MRL/lpr mice and analyzing pristane-treated LCN2-/- mice.

Results: LCN2 is highly expressed in CD4+ T cells and in renal tissues, and is associated with severe renal damage in patients with LN and in mice with experimental lupus. LCN2 promotes IFN-γ overexpression in CD4+ T cells through the IL-12/STAT4 pathway in an autocrine or paracrine manner. Both neutralization of LCN2 in MRL/lpr mice and genetic depletion of LCN2 in pristane-induced lupus mice greatly ameliorate nephritis. The frequency and number of splenic and renal Th1 cells decrease in proportion to LN disease activity. Conversely, administration of LCN2 exacerbates the disease with significantly higher renal activity scores and increased numbers of Th1 cells.

Conclusions: LCN2 plays a crucial role in Th1 cell differentiation, and may present a potential therapeutic target for LN.

Keywords: Th1 cells; lipocalin-2; lupus nephritis.

Copyright © 2020 by the American Society of Nephrology.

Figures

Figure 1.

LCN2 expression is elevated in CD4+ T cells and the kidneys of patients with LN. (A) Screening of the top 20 upregulated genes in PBMCs from patients with SLE (SLE; n=3) and healthy controls (C; n=3). (B) Real-time PCR validation of gene expressions in PBMCs from patients with SLE (n=6) and healthy control (HCs; n=6). (C) Expression of LCN2 in naive CD4+ T cells derived from patients with SLE and LN (n=14), patients without LN (non LN, n=16), and HCs (n=20). (D) Immunochemical staining of LCN2 in the kidneys from patients with class 2 LN (n=5), class 3 LN (n=5), class 4 LN (n=8), class 4+5 (n=3), and class 5 (n=4), respectively. Quantification of LCN2-positive area is shown in the right panel. (E) The data plots show the correlation analysis of LCN2-positive area in kidneys with the active index, chronic index, and interstitial inflammation in patients with LN (n=25). P values are determined by Mann–Whitney U test in (B), one-way ANOVA with Tukey multiple comparisons test in (C), and one-way ANOVA with Dunnett multiple comparisons test in (D). Both correlation coefficient r and P values are calculated by the Spearman r test in (E). Data are shown as mean±SEM. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. Original magnification, ×100. Ctrl, control.

Figure 2.

LCN2 expression is increased in T cells, macrophages, neutrophils, and renal TECs in LN kidneys. Representative photos of immunofluorescence staining of LCN2 and different cell type markers in renal biopsy sections of controls (Ctrl), patients with LN, and those with DN. (A) LCN2+ cells (green) and CD3 (red) are stained. (B) LCN2+ cells (green) and CD68 (red) are stained. (C) LCN2+ cells (green) and CD15 (red) are stained. (D) LCN2+ cells (green) and E-cadherin (red) are stained. Colocalization is visualized by the yellow merge of red and green signals. Nuclei are stained with 4′,6-diamidino-2-phenylindole (blue). (E) Quantification of LCN2 immunofluorescence intensity in the experiments of (A–D) (n=6). One-way ANOVA with Dunnett multiple comparisons test is used. Data are shown as mean±SEM. *P<0.05, **P<0.01. Original magnification, ×100. Mφ, macrophages; Neu, neutrophils; T, T cells; TEC, tubular epithelial cells.

Figure 3.

LCN2 accelerates the development of LN. (A) Albuminuria is expressed as micrograms of albumin per milligram of urinary creatinine during the course of treatments in LCN2- (n=6) or PBS- (n=7) treated mice. (B) Spleen/body weight ratio of both groups. (C) Immunofluorescence staining for mouse IgG and C3. Original magnification, ×400. (D) Representative photographs of kidney sections stained with hematoxylin and eosin (HE) and Periodic acid–Schiff (PAS). Original magnification, ×100. Quantification of glomerular, tubulointerstitial, and perivascular pathology. (E) The mRNA expression of IFN-γ, IL-6, TNFα, IL-1β, and MCP-1 in the kidneys. (F) Representative photographs of CD68 and Gr-1–stained kidney sections from LCN2- or PBS-treated mice. Original magnification, ×400. Quantification of positive cells per high-power field (HPF) is shown in the right panels. (G) Representative flow cytometric analysis of Th1 cells in spleens and lymph nodes from mice treated with LCN2 or PBS. The percentage and number of Th1 cells are shown in the right two panels, respectively. Data are representative of two independent experiments. P values are determined by two-tailed unpaired t test in (A–D, F, and G) and by Mann–Whitney U test in (E). Data are shown as mean±SEM. *P<0.05, **P<0.01, ***P<0.001.

Figure 4.

LCN2 deficiency prevents LN development by suppressing Th1 cells. (A) Percentage survival, showing animals euthanized due to pristane treatment (n=6 for WT or LCN2−/− mice, n=20 for WT+pristane or LCN2−/−+pristane mice). (B) The spleen/body weight ratio from PBS-treated WT (n=6) and LCN2−/− mice (n=6) and pristane-treated WT (n=14) and LCN2−/− mice (n=20). (C) Albuminuria and (D) serum creatinine levels from each treatment group. (E) Representative photographs of kidney sections stained with hematoxylin and eosin (HE) and Periodic acid–Schiff (PAS). Original magnification, ×100. Quantification of glomerular, tubulointerstitial, and perivascular pathology. (F) Photos and graphs of IgG and C3 deposition in glomeruli. Original magnification, ×400. (G and H) Representative flow cytometric analysis of Th1 cells in (G) splenic and (H) renal (n=4 for each group) CD4+ T cells. The percentage and number of Th1 cells are shown in the right two panels, respectively. Data are representative of three independent experiments. P values are determined by one-way ANOVA with Dunnett multiple comparisons test in (B–D, G, and H) and by two-tailed unpaired t test in (E and F). Data are shown as mean±SEM. *P<0.05, **P<0.01, ***P<0.001.

Figure 5.

Administration of anti-LCN2 antibodies ameliorates the pathologic phenotype of lupus mice. (A) Percentage survival of mice treated with anti-LCN2 (n=8) or control antibodies (n=10). (B) Albuminuria during the course of treatments in mice treated with anti-LCN2 or control antibodies. (C) Spleen/body weight ratio in mice treated with anti-LCN2 (n=8) or control antibodies (n=8). (D) Representative photographs of kidney sections stained with hematoxylin and eosin (HE) and Periodic acid–Schiff (PAS). Original magnification, ×100. Quantification of glomerular, tubulointerstitial, and perivascular pathology. (E) Photos and graphs of IgG and C3 deposition in glomeruli. Original magnification, ×400. (F) Representative photographs of CD68- and Gr-1–stained kidney sections. Original magnification, ×400. Quantification of positive cells per high-power field (HPF) is shown in the right two panels. (G) The mRNA expression of IFN-γ, IL-6, TNFα, IL-1β, and MCP-1 in the kidneys. (H) Representative flow cytometric analysis of Th1 cells in the spleens and lymph nodes from mice treated with anti-LCN2 or control antibodies. The percentage and number of Th1 cells are shown in the right two panels, respectively. Data are representative of two independent experiments. P values are determined by two-tailed unpaired t test in (B–E), the top scatter diagram in (F), IFN-γ, IL-6, and MCP-1 in (G), and the percentage of Th1 cells in (H); and by Mann–Whitney U test in the lower scatter diagram in (F), TNFα and IL-1β in (G), and the number of Th1 cells in (H). Data are shown as mean±SEM. *P<0.05, **P<0.01, ***P<0.001.

Figure 6.

LCN2 deficiency suppresses STAT4-mediated Th1 cell differentiation in vitro. (A) Representative CD4 and IFN-γ staining profile in isolated T cells from WT and LCN2−/− mice with/without recombinant LCN2 (rhLCN2) under Th1 cell–polarizing conditions. The percentage of Th1 cells is shown in the right panel. n=5 per group. (B) Protein levels of IFN-γ (n=5) in cultured supernatants of CD4+ T cells from WT and LCN2−/− mice with/without rhLCN2 under Th1 cell–polarizing conditions for 5 days. (C) Protein level of 24p3R in splenic naive CD4+ T cells (n=2). (D) Western blot analysis of the time course of phosphorylated-STAT4 (p-STAT4) expression in anti-CD3/CD28 antibody–treated CD4+ T cells from WT and LCN2−/− mice with/without rhLCN2 after exposure to IL-12. p-STAT4 expression is normalized to STAT4. Time course of p-STAT4 expression is shown in the right panel. Data are representative of three independent experiments. (E) mRNA expression for IL12Rβ2, T-bet, and IFN-γ (n=5) of CD4+ T cells from WT and LCN2−/− mice under Th1 cell–polarizing conditions. mRNA levels were normalized to the expression of glyceraldehyde 3-phosphate dehydrogenase (GAPDH). (F) Representative CD4 and IFN-γ staining profile in isolated T cells from MRL/lpr mice adding anti-LCN2 or control antibodies under Th1 cell–polarizing conditions. The percentage of Th1 cells is shown in the right panel. n=5 per group. P values are determined by one-way ANOVA with Dunnett multiple comparisons in (A, B, and D), and by Mann–Whitney U test in (E and F). Data are shown as mean±SEM. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

Figure 7.

Schematic diagram showing the contribution of LCN2 to LN. LCN2 promotes Th1 cell differentiation via the IL-12/STAT4 pathway, leading to the exacerbation of LN. During pathologic exacerbation of LN, the kidney (TECs and infiltrating leukocytes) can also serve as the source of increased LCN2, forming a positive feedback loop. Mφ, macrophages; Neu, neutrophils; TEC, tubular epithelial cells; T, T cells; P, phosphorylated.