Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2016 Sep;68(9):2244-56.
doi: 10.1002/art.39673.

Inhibition of G Protein βγ Subunit Signaling Abrogates Nephritis in Lupus-Prone Mice

Affiliations
Free PMC article

Inhibition of G Protein βγ Subunit Signaling Abrogates Nephritis in Lupus-Prone Mice

Javier Rangel-Moreno et al. Arthritis Rheumatol. .
Free PMC article

Abstract

Objective: Despite considerable advances in the understanding of systemic lupus erythematosus (SLE), there is still an urgent need for new and more targeted treatment approaches. We previously demonstrated that small-molecule blockade of G protein βγ subunit (Gβγ) signaling inhibits acute inflammation through inhibition of chemokine receptor signal transduction. We undertook this study to determine whether inhibition of Gβγ signaling ameliorates disease in a mouse model of SLE.

Methods: Lupus-prone (NZB × NZW)F1 female mice were prophylactically or therapeutically treated with the small-molecule Gβγ inhibitor gallein. Tissue samples were analyzed by flow cytometry and immunohistochemistry. The development and extent of nephritis were assessed by monitoring proteinuria and by immunohistochemical analysis. Serum immunoglobulin levels were measured by enzyme-linked immunosorbent assay, and total IgG and anti-double-stranded DNA (anti-dsDNA) antibody-secreting cells were measured by enzyme-linked immunospot assay.

Results: Gallein inhibited accumulation of T cells and germinal center (GC) B cells in the spleen. Both prophylactic and therapeutic treatment reduced GC size, decreased antibody-secreting cell production in the spleen, and markedly decreased accumulation of autoreactive anti-dsDNA antibody-secreting cells in kidneys. Gallein also reduced immune complex deposition in kidneys. Finally, gallein treatment dramatically inhibited kidney inflammation, prevented glomerular damage, and decreased proteinuria. Mechanistically, gallein inhibited immune cell migration and signaling in response to chemokines in vitro, which suggests that its mechanisms of action in vivo are inhibition of migration of immune cells to sites of inflammation and inhibition of immune cell maturation.

Conclusion: Overall, these data demonstrate the potential use of gallein or novel inhibitors of Gβγ signaling in SLE treatment.

Figures

Figure 1
Figure 1. Gallein inhibits migration of mouse neutrophils and human T cells in response to GPCR-coupled chemoattractants
A) Neutrophils were isolated from the bone marrow and pretreated with 10 μΜ gallein for 10 minutes and allowed to migrate in response to either 250 nM fMLP or 100 ng/mL GM-CSF. The filter was stained and cells attached to the bottom were counted. Background migration in response to vehicle was subtracted from migration in response to chemoattractant. B) Gallein inhibits migration of human T cells in response to CXCL12. Human T cells were pretreated with 10 μM gallein for 10 minutes and allowed to migrate in response to 100ng/mL CXCL12 for 3 hours, 37°C. The filter was stained and cells attached to bottom were counted. Background migration was subtracted. C) Gallein inhibits membrane PIP3 production in response to fMLP in differentiated HL-60 cells. Differentiated HL-60 cells stably expressing PH-AKT GFP were pretreated with 10 μΜ gallein for 10 minutes prior to stimulation with 250 nM fMLP. Images were taken every 10 sec and the relative fluorescence was determined by using the ratio of membrane over cytoplasmic fluorescence over time. Three cells were averaged per experiment and each experiment repeated three times. Data is expressed as mean ± standard error of the mean. Statistical significance was analyzed with a Student’s T-test: *, p<0.05, ***, p<0.001, ****, p<0.0001.
Figure 2
Figure 2. Prophylactic administration of gallein blocks lymphocyte recruitment and impairs germinal center responses in the spleen of lupus prone mice
Groups of 18 week old NZB/NZW female mice were treated with vehicle, 20 mg/kg or 35 mg/kg gallein for 20 weeks (n=8 mice/group, 3 times a week intraperitoneal administration). A) At the end of the therapeutic regimen, spleens were collected and different cell populations were enumerated by flow cytometry. Total numbers of CD3+CD4+CD44CD62+ naïve-, CD3+CD4+CD44+CD62L effector- and CD3+CD4+CD44+CD62L+ central memory CD4 T cells are shown in top panels. Numbers of CD3+CD4+CXCR5+ICOS+PD1+ T follicular helper cells and CD19+PNA+FAS+ germinal center B cells are depicted in bottom panels. B) Spleens were collected at the end of the prophylactic therapy and embedded in OCT. 5 μm frozen sections were stained with antibodies against IgD (red) and IgG (white), in combination with fluorescein isothiocyanate-labeled peanut agglutinin (Green). Representative pictures taken at 200 x magnification are shown. C) All PNA+ germinal centers in individual spleen frozen sections (n = 8 spleen sections per group) were outlined with an automated tool of the Zeiss microscope to calculate their size (left panel), number (center panel) and percentage covered by germinal centers per spleen section (right panel). Representative results from two independent experiments with similar results are shown. Data is expressed as mean ± standard error of the mean. Statistical significance was calculated by non-parametric Mann Whitney test. *, p<0.05, **, p<0.005, ****, p<0.0001.
Figure 3
Figure 3. Gallein decreases accumulation of auto-reactive antibody secreting cells in lupus prone mice
Mice were treated as in figure 2. Spleen, BM and kidney were collected to isolate leukocytes. A) White cells were added to ELISpot plates coated with anti IgG or double stranded DNA (dsDNA) to enumerate isotype-switched (top panels) and dsDNA specific antibody secreting cells (ASC) (bottom panels) in different organs. n=8 mice/group B) Gallein therapy does not significantly reduce serum levels of dsDNA IgG antibodies. n=8 mice/group C) Prophylactic administration of gallein decreased IgG deposition in the kidneys of lupus prone mice. Representative pictures were taken at a 200 x magnification (n= 8 kidney sections/group). The area of IgG deposition was measured with Image J in a blinded manner. Representative results from two independent experiments with similar results are shown. Data is expressed as mean ± standard error of the mean. Statistical significance was calculated by non-parametric Mann Whitney test. *, p<0.05, ***, p<0.001.
Figure 4
Figure 4. Inhibition of G protein βγ signaling prevents accumulation of immune cells in inflamed kidneys at early stages of experimental lupus
After 20 weeks of gallein administration kidneys were fixed in formalin and embedded in paraffin. A) 5 μm kidney sections were stained with hematoxylin and eosin. Representative 200 x magnification pictures from kidneys showing inflammatory cell infiltrates around blood vessels are shown. Gross histological findings were confirmed by measurement of all perivascular cell infiltrates (right panel, n=8 mice/group). B) 5 μm kidney sections were stained with hematoxylin and eosin. Representative 200 x magnification pictures from kidneys showing glomerular inflammation are shown. Right panel shows quantitative blinded evaluation of glomerular inflammation by a certified pathologist (right graph) in individual kidney sections (n = 8 kidney sections/group). C) Effects of gallein treatment on accumulation of CD3+ and B220+ cells around blood vessels in the kidneys of lupus prone mice. 5 μm kidney sections were stained with primary antibodies against CD3 (red), IgG (green) and B220 (white). Representative 200 x magnification pictures from two independent experiments with similar results are shown. D) Protein levels in the urine of mice were monitored. n=8 mice/group Data is expressed as mean ± standard error of the mean. Statistical significance was calculated by non-parametric Mann Whitney test. **, p=<0.005, ***, p<0.0005, ****, p<0.0001.
Figure 5
Figure 5. Germinal centers and auto-reactive ASC are major targets of gallein in mice with active experimental lupus
Groups of 28 weeks old NZB/NZW female mice, with established proteinuria, were treated with vehicle or 35 mg/kg gallein (n=8/group, 5 times a week administration). A) At the end of the therapy, mice were sacrificed and spleens were fixed in neutral buffered formalin and embedded in paraffin. 5 μm paraffin spleen sections were stained with FITC-labeled peanut agglutinin, in combination with primary antibodies against proliferating cell nuclear antigen (red) and B220 (white). 200 x magnification pictures show germinal centers (GC) that are outlined with a yellow dashed line. Morphometric analysis is shown on the right. B) CD19+kappalight+MHC CII+ plasmablasts and CD19+Kappalight+MHC CII plasma cells were enumerated in red blood cell-free cell suspensions by flow cytometry. C) Spleen, BM and kidney were collected to isolate leukocytes. White cells were added to ELISpot plates coated with anti IgG or double stranded DNA (dsDNA) to enumerate isotype-switched and dsDNA specific antibody secreting cells (ASC) in different organs. Representative results from two independent experiments with similar results are shown. Data is expressed as mean ± standard error of the mean. Statistical significance was calculated by non-parametric Mann Whitney test. *, p<0.05, **, p=<0.005, ****, p<0.0001.
Figure 6
Figure 6. Gallein halts deterioration of kidney function in mice with active lupus
NZB/NZW mice with established lupus were treated as in figure 5. A) Kidneys were fixed in neutral buffered formalin and embedded in paraffin and 5 μm kidney sections were stained with hematoxylin and eosin. Representative 200 x magnification pictures from kidneys showing inflammatory cell infiltration around blood vessels and the effects of gallein treatment are shown. Gross histological findings were confirmed by measurement of all perivascular cell infiltrates (right panel). Glomerular scores were: Cntl: 2±0.29 n=3, Gallein: 1±0.29 n=6, these values were not statistically different. B) Effects of gallein treatment on accumulation of CD3+ and B220+ cells around blood vessels in the kidneys of lupus prone mice. 5 μm kidney sections were stained with primary antibodies against CD3 (red), IgG (green) and B220 (white), followed by detection with fluorescently-labeled secondary antibodies. Representative 200 x magnification pictures from two independent experiments with similar results are shown. Quantitation of IgG deposition in 8 kidney sections for each group are shown on the right. C) Proteinuria score at different time-points after treatment of NZB/NZW mice with established lupus. Arrow indicates the beginning of the treatment. n=8 mice/group. Data is expressed as mean ± standard error of the mean. Statistical significance was calculated by non-parametric Mann Whitney test. *, p<0.05; **, p=<0.005.

Similar articles

See all similar articles

Cited by 8 articles

See all "Cited by" articles

Publication types

MeSH terms

Feedback