Centrally Acting Angiotensin-Converting Enzyme Inhibitor Suppresses Type I Interferon Responses and Decreases Inflammation in the Periphery and the CNS in Lupus-Prone Mice

Abstract

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by multi-organ damage. Neuropsychiatric lupus (NPSLE) is one of the most common manifestations of human SLE, often causing depression. Interferon-α (IFNα) is a central mediator in disease pathogenesis. Administration of IFNα to patients with chronic viral infections or cancers causes depressive symptoms. Angiotensin-converting enzyme (ACE) is part of the kallikrein-kinin/renin-angiotensin (KKS/RAS) system that regulates many physiological processes, including inflammation, and brain functions. It is known that ACE degrades bradykinin (BK) into inactive peptides. We have previously shown in an in vitro model of mouse bone-marrow-derived dendritic cells (BMDC) and human peripheral blood mononuclear cells that captopril (a centrally acting ACE inhibitor-ACEi) suppressed Type I IFN responsive gene (IRG) expression. In this report, we used the MRL/lpr lupus-prone mouse model, an established model to study NPSLE, to determine the in vivo effects of captopril on Type I IFN and associated immune responses in the periphery and brain and effects on behavior. Administering captopril to MRL/lpr mice decreased expression of IRGs in brain, spleen and kidney, decreased circulating and tissue IFNα levels, decreased microglial activation (IBA-1 expression) and reduced depressive-like behavior. Serotonin levels that are decreased in depression were increased by captopril treatment. Captopril also reduced autoantibody levels in plasma and immune complex deposition in kidney and brain. Thus, ACEi's may have potential for therapeutic use for systemic and NPSLE.

Keywords: angiotensin-converting enzyme inhibitor; captopril; depression; immune complex; lupus-prone mice; neuroinflammation; serotonin; type I interferon.

FIGURE 1

Young MRL/lpr mice present a constitutive IFN signature. (A) Gene expression of IRF7, CXCL10, and IFNγ in brain and kidney was analyzed by Taqman qPCR. ΔΔCt method was used to calculate the fold changes using MRl/wt mice as controls. Cyclophilin gene was used for normalizing gene expression. (B) Multiplex cytokine analysis was done using the MSD ELISA kit. Key cytokines >5 pg/ml that are significantly different between the MRL/lpr and MRL/wt mice are represented. (C) Plasma IFNα levels were measured using high sensitivity Verikine ELISA kit from PBL. (D) Serotonin levels were measured in the plasma; levels in MRL/lpr were significantly low. Unpaired t test statistical analysis was performed and p < 0.05 was considered significant; *p < 0.05, **p < 0.01, and ****p < 0.0001; n = 3–5 mice/group.

FIGURE 2

Captopril reduces IRGs in brain, spleen and kidney in lupus-prone mice. (A) MRL/lpr mice were treated with captopril (i.p. 5 mg/kg body wt.) for 2 weeks and analyzed for IRGs in spleen, kidney and brain by Taqman qPCR. IRG expression in the spleen, kidney and brain was analyzed after oral captopril treatment in MRL/lpr (B) and NZB/W F1 mice (C). ΔΔCt method was used to calculate the fold changes in gene expression using PBS-treated mice as a control group. Cyclophilin gene was used for normalizing gene expression. N = 3–5 mice/group; Unpaired t test statistical analysis was performed and p < 0.05 was considered significant; *p < 0.05, **p < 0.01, and ***p < 0.001.

FIGURE 3

Captopril reduces IFN responsive protein expression. Plasma from MRL/lpr mice induced with exogenous IFNα and treated with captopril for 2 weeks were analyzed for cytokines and chemokines using MSD multiplex ELISA kit. Significantly different cytokines and chemokines are represented, (A) >10 pg/ml and (B) <10 pg/ml (n = 4/group). (C) Serotonin levels in plasma of IFNα induced MRL/lpr mice was measured using ELISA. (D) Splenocytes of IFNα treated MRL/lpr mice were harvested, stained for intracellular IFNα, gated and analyzed on CD11c + cells. Median fluorescence intensities (MFI) are represented as histograms on the left and the corresponding colors for the different groups indicated in the bar graph on the right (n = 4/group). Unstained control is shown in gray in the histogram. (E) Spleen lysates of exogenous IFNα treated MRL/lpr mice were analyzed by western blotting to detect IFNAR1 protein. Actin was used a loading control. Band intensities were analyzed using ImageJ software and normalized values are represented in the graph. Unpaired t test statistical analysis was performed and p < 0.05 was considered significant; *p < 0.05, **p < 0.01, and ***p < 0.001.

FIGURE 4

Captopril reduces microglial activation in the brain. Young MRL/lpr mice treated with captopril (i.p.) were perfused at end of treatment and brain tissue was harvested, paraffin embedded and analyzed for IBA-1 expression by chromagen (DAB) staining and IBA-1 staining in the PBS and captopril treated groups. IBA-1 staining of brain microglia shows more activation in PBS-treated MRL/lpr mice than in captopril-treated mice. Inset shows 40x magnification of the cortex region showing activated microglia with retracted processes and bigger cell bodies in PBS- treated mice vs. less activated microglia in captopril-treated mice. Cells stained for IBA-1 (brown) were counted in each of 6 fields on each slide for each mouse and averaged; n = 3–5 mice/group. Slides were coded and read in a blinded fashion to avoid any bias in interpretation. Unpaired t test statistical analysis was performed and p < 0.05 was considered significant; *p < 0.05.

FIGURE 5

Captopril suppresses depression-like and anxiety-like behavior in lupus-prone mice. The forced swim test was performed in MRL/lpr mice after systemic captopril administration (A,B) and oral captopril administration (C,D) as described in the Methods section. Percent time immobile is represented in (A,C). Average latency to immobility (in seconds) is represented in (B,D). Captopril mice showed shorter time of immobility and longer latency indicating less depressive behavior than the untreated controls. Anxiety-like behavior was measured by EPM in the MRL/lpr (E) and NZB/W F1 (F) mice. Percent time spent in the open arms is shown. Captopril treated NZB/W F1 mice spent more time in open arms indicating less anxious behavior compared to PBS treated controls. Data are shown as mean + SE. Students t test *p < 0.05, n = 6/group.

FIGURE 6

Oral captopril reduces BUN and autoantibody levels in lupus-prone mice. (A) MRL/lpr mice orally treated with captopril for about a month and blood was assessed for BUN using Bayer’s dipstick method as per manufacturer’s protocol and scored as 1 (5–15 mg/dL), 2 (15–26 mg/dL), 3 (30–40 mg/dL), and 4 (50–80 mg/dL), as indicated by the color intensity. (B) Proteinuria was analyzed using the Multistix dipstick method and color intensities were scored as per manufacturer’s instructions 1-; 2-; 3-; 4. Captopril-treated MRL/lpr (n = 5/group); (C) and NZB/W F1 (n = 8/group); (D) mice were analyzed for anti-dsDNA and anti-chromatin autoantibodies. O.D.’s were plotted and compared between the treated and untreated groups. The p values shown in green are comparisons before and after treatment in that group using paired Student’s t test. *p < 0.05 and **p < 0.01.

FIGURE 7

Oral captopril reduces IgG and complement deposition in the kidney. Frozen kidney sections from MRL/lpr mice treated with oral captopril were analyzed for C3 complement and IgG deposition by immunofluorescence staining. (A) 200X magnified image shows glomeruli (indicated by arrows) stained more intense with C3 or IgG in the untreated (PBS) group as compared to less intense staining in the captopril-treated group. (B) Numerical values of stain intensities are represented in the bar graphs (n = 5/group). Unpaired t test statistical analysis was performed and p < 0.05 was considered significant; *p < 0.05.

FIGURE 8

Oral captopril reduces IgG deposition in the brain. Frozen brains from captopril- treated and control MRL/lpr mice were sagitally sectioned and stained for IgG (FITC), tomato lectin (Texas red) for microvessels and DAPI and analyzed in the cortex and hippocampal regions. Representative images (400x magnification) show increased staining for IgG (indicated by arrows) in the hippocampal region of untreated mice than captopril-treated mice (n = 5/group). Staining intensity was quantified by ImageJ and unpaired t statistical analysis was performed, *p < 0.05.