Lipocalin-2 drives neuropsychiatric and cutaneous disease in MRL/lpr mice

Abstract

Introduction: Approximately 20-40% of patients with systemic lupus erythematosus (SLE) experience neuropsychiatric SLE (NPSLE), which often manifests as cognitive dysfunction and depression. Currently, there are no approved treatments for NPSLE because its underlying mechanisms are unclear. Identifying relevant mediators and understanding their contribution to pathogenesis are crucial for developing targeted treatment options. Lipocalin 2 (LCN2) is a multifunctional acute-phase protein that plays important roles in immune cell differentiation, migration, and function. LCN2 has been implicated in models of neuroinflammatory disease.

Methods: We generated an LCN2-deficient MRL/lpr mouse to evaluate the effects of LCN2 on this classic NPSLE model. To evaluate the effects of LCN2 deficiency on behavior, the mice underwent a battery of behavioral tests evaluating depression, memory, and anxiety. Flow cytometry was used to quantify immune cell populations in the brain, blood, and secondary lymphoid organs. Cutaneous disease was quantified by scoring lesional skin, and skin infiltrates were quantified through immunofluorescent staining. Systemic disease was evaluated through measuring anti-nuclear antibodies by ELISA.

Results: In this study, we found that LCN2 deficiency significantly attenuates neuropsychiatric and cutaneous disease in MRL/lpr lupus prone mice, likely by decreasing local infiltration of immune cells into the brain and skin and reducing astrocyte activation in the hippocampus. Anti-nuclear antibodies and kidney disease were not affected by LCN2.

Discussion: As there was no effect on systemic disease, our results suggest that the inflammatory effects of LCN2 were localized to the skin and brain in this model. This study further establishes LCN2 as a potential target to ameliorate organ injury in SLE, including neuropsychiatric and cutaneous disease.

Keywords: MRL/lpr; cutaneous systemic lupus erythematosus; lipocalin-2; neuropsychiatric lupus; systemic lupus erythematosus.

Figure 1

LCN2 deficiency improves behavioral abnormalities in MRL/lpr mice. (A, B) LCN2 deficient mice showed significant improvement in depression-like behavior in the Porsolt swim and the tail suspension tests. (C) LCN2-deficient mice showed improvement in spatial memory during the object placement task. (D) Pattern recognition memory was unaffected in LCN2-deficient mice as displayed in the object recognition task. (E) Locomotion was not different between groups as quantified through the behavioral spectrometer. (F) Behavioral spectrometry showed no differences in generalized activity between the strains. For all panels: n.s., not significant; *p<0.05, **p<0.01, and ***p<0.001.

Figure 2

LCN2 deficiency decreases CP infiltrates. (A) Representative H&E image of CP infiltration in an LCN2-KO mouse. (B) Representative H&E image of the CP in an MRL/lpr mouse. (C) LCN2-KO mice have lower CP infiltrates when scored quantitatively. (D) CP C3 and (E) IgG expression were unaffected by LCN2 deficiency. (F) Albumin leakage in CP was unchanged in LCN2-deficient mice compared to MRL/lpr mice. For all panels: n.s., not significant and *p<0.05.

Figure 3

LCN2 deficiency decreases immune cell counts in MRL/lpr brains. Cell counts were calculated per whole brain. (A-I) B cells, plasma cells, total T cells, helper T cells (CD4+), CD138+ helper T cells, cytotoxic T cells, CD138+ cytotoxic T cells, inflammatory monocytes, and dendritic cells respectively were all significantly decreased in the brains of LCN2-deficient as compared to wild-type (LCN2 sufficient) MRL/lpr mice. For all panels: *p<0.05 and **p<0.01.

Figure 4

LCN2 does not mitigate systemic autoimmunity and inflammation. LCN2 has no effect on serum autoantibodies including (A–C) anti-chromatin IgG, anti-DNA IgG, and anti-histone IgG. No effect is seen in circulating PBMC numbers including (D-L) B cells, plasma cells, T cells, helper T cells, cytotoxic T cells, CD138+ helper T cells, cytotoxic T cells, CD138+ cytotoxic T cells, inflammatory monocytes, and dendritic cells respectively. For all panels: **p<0.01 and ****p<0.0001.

Figure 5

LCN2 deficiency does not affect spleen cell subsets. The number of immune cells in the spleen was not different between MRL/lpr and LCN2-KO MRL/lpr mice. (A-I) B cells, plasma cells, T cells, helper T cells, cytotoxic T cells, CD138+ helper T cells, cytotoxic T cells, CD138+ cytotoxic T cells, inflammatory monocytes, and dendritic cells, respectively. None of the panels depict significant differences.

Figure 6

LCN2 deficiency improves cutaneous disease in MRL/lpr mice. (A) A representative image of lesional skin on MRL/lpr mice. Erythema, alopecia, and skin thickening are apparent. (B) shows a markedly improved skin phenotype in LCN2-KO mice. (C) Quantitative skin scores (recorded blindly) confirmed the significant improvement in skin disease in LCN2-KO mice. (D) T cells were decreased in the skin of LCN2-deficient mice. (E) Representative IF images of skin from LCN2-KO MRL/lpr stained for T cells. (F) Representative IF image of T cells in MRL/lpr lesional skin. (G) Neutrophils were decreased in the skin of LCN2-KO mice. (H) Representative image of cutaneous neutrophils in LCN2-KO mice. (I) Representative IF image of cutaneous neutrophils in MRL/lpr mice. (J) LCN2 deficiency reduced skin macrophage infiltrates. (K) Representative IF image of LCN2-KO skin macrophage infiltrates. (L) MRL/lpr skin macrophage infiltrates. For all panels: *p<0.05, **p<0.01.

Figure 7

LCN2 promotes an inflammatory astrocyte phenotype. Cortical astrocytes isolated from newborn MRL/lpr pups were evaluated by immunofluorescence for the presence of activation markers following different inflammatory stimuli. LCN2 was added at a concentration of 25 μg/mL, LPS at a concentration of 100 ng/mL, and IFNγ was added at a concentration of 1 μg/mL. Treatment duration for each condition was 10 hours. (A) Representative images of treated astrocytes following each stimulus. Vertically listed are the different treatment conditions, while horizontally listed are the different activation markers being measured in each column. The last column in this panel shows the overlay of all 3 markers. (B) C3 protein expression is induced in LCN2 treated primary astrocytes. (C) LCN2 induces GFAP expression in primary astrocytes. (D) S100B, another astrocytic inflammatory marker, is also increased upon LCN2 exposure. For all panels: *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001.