CSF-1R inhibition attenuates renal and neuropsychiatric disease in murine lupus

Abstract

Systemic lupus erythematosus (SLE) is a multifactorial autoimmune disease that can affect multiple end organs. Kidney and brain are two of the organs most commonly involved in SLE. Past studies have suggested the importance of macrophages in the pathogenesis of lupus nephritis (LN). Furthermore, as the immune effectors of the brain, microglia have been implicated in pathways leading to neuropsychiatric SLE (NPSLE). We depleted macrophages and microglia using GW2580, a small colony stimulating factor-1 receptor (CSF-1R) kinase inhibitor, in MRL-lpr/lpr (MRL/lpr) mice, a classic murine lupus model that displays features of both LN and NPSLE. Treatment was initiated before the onset of disease, and mice were followed for the development of LN and neurobehavioral dysfunction throughout the study. Treatment with GW2580 significantly ameliorated kidney disease, as evidenced by decreased proteinuria, BUN, and improved renal histopathology, despite equivalent levels of IgG and C3 deposition in the kidneys of treated and control mice. We were able to confirm macrophage depletion within the kidney via IBA-1 staining. Furthermore, we observed specific improvement in the depression-like behavioral deficit of MRL/lpr mice with GW2580 treatment. Circulating antibody and autoantibody levels were, however, not affected. These results provide additional support for the role of macrophages as a potentially valuable therapeutic target in SLE. Inhibiting CSF-1 receptor signaling would be more targeted than current immunosuppressive therapies, and may hold promise for the treatment of renal and neuropsychiatric end organ disease manifestations.

Keywords: CSF-1R; Lupus nephritis; Macrophages; Neuropsychiatric lupus; SLE.

Fig. 1

Renal function in GW2580 treated mice. Proteinuria levels were measured over time via uristix (A). Urine albumin levels were measured via ELISA and normalized to urine creatinine levels to adjust for varying volumes of urinary output (B). BUN was measured via ELISA to assess kidney function (C). (GW2580 treated, n=9; Control treated, n=10) (*p<0.05, **p<0.01).

Fig. 2

Renal histopathology. The left side of panel A shows representative images of control treated mice with interstitial inflammation (arrows, top left), deposits (arrow) with endocapillary proliferation (*) (middle left), and necrotizing crescent (arrow, bottom left). GW2580 treated mice displayed improved histopathology, although still displayed patchy interstitial inflammation (arrow, top right), mesangial deposits (arrow, middle right), and less extensive endocapillary proliferation (arrow, bottom right). Scores for crescents, endocapillary proliferation, and interstitial inflammation are shown in B and C. (GW2580 treated, n=9; Control treated, n=10) (*p<0.05).

Fig. 3

Renal IgG and C3 deposition. Kidney sections were stained for IgG deposition (green) and C3 deposition (red). Co-staining of both IgG and C3 appears in yellow. Both control treated and GW2580 treated mice showed similar levels of deposition of both IgG and C3, as seen in these representative images (GW2580 treated, n=6; Control treated, n=6).

Fig. 4

IBA-1+ macrophages in the kidney. Kidney sections were stained with the macrophage marker IBA-1 to assess macrophage numbers within the kidney. (A) Control treated mice have more numerous IBA-1+ cells than GW2580 treated mice. Glomeruli are circled in yellow, and are infiltrated by macrophages in the control treated mice (short arrows). Additionally, periglomerular accumulation of macrophages can also be seen in control treated mice (long arrows) (GW2580 treated, n=9; Control treated, n=10) (**p<0.01). Intraglomerular macrophages were quantified in (B) (GW2580 treated, n=7, 73 total glomeruli counted; Control treated, n=9, 91 total glomeruli counted).

Fig. 5

Serum antibody and autoantibody levels. Total IgG levels were measured in the serum from 18 week old mice (A). Additionally, anti-DNA antibodies (B) and anti-chromatin antibodies (C) were measured in serum from the same time point. Shown is the O.D. normalized to healthy mouse serum. (GW2580 treated, n=9; Control treated, n = 10)

Fig. 6

GW2580 treated mice display ameliorated depression-like behavior and increased microglial activation. (A) Depression-like behavior was assessed in the Porsolt swim test for 3 minutes. (B) Representative images of IBA-1 staining in the cortex from the PBS and GW2580 treated groups is shown, along with the intensity of IBA-1 staining as measured by ImageJ. (C) Representative images of H&E staining in the third ventricles of PBS and GW2580 treated mice. (D) Representative images of albumin staining are shown, with arrows indicating the blood vessels. The number of animals in the forced swim test in the PBS and GW2580 treated groups is 10 and 10, respectively. For IBA-1, H&E, and albumin staining, the number is 10 and 9, respectively.

Fig. 7

GW2580 treatment modulates cytokine expression. GW2580 treated mice have significantly decreased concentrations of several inflammatory cytokines as compared to control treated mice in both kidneys (A) and brain (B) (GW2580 treated, n=4; Control treated, n = 4) (*p<0.05).