Figure 1.
Neutrophils and dendritic cells contribute to elevated BAFF levels in the absence of BCMA.
(A) BAFF levels were measured in sera from mice of the indicated genotype. Each symbol represents a single animal. (B) Spleen sections from 6 month-old mice were stained with H&E. Each arrow represents a neutrophil identified by nuclear structure, with the boxed neutrophil magnified 100x in the inset image. Shown are representative images from 5 mice per genotype. RP – red pulp, FO – follicle, MZ – mantle zone. (C) Left panels: spleen sections from 6-month-old mice were analyzed for BAFF-producing cells by immunofluorescence. BAFF – green, CD11c or Ly6G – red. Scale bar represents 50 µm. Arrows represent cells with co-localization of BAFF and either CD11c or Ly6G, with the boxed cell magnified in the inset images. One representative image from 4 mice of each genotype is shown. Right panel: quantification of the intensity of BAFF staining from spleen-resident CD11c+ DCs and Ly6G+ neutrophils was measured by CellProfiler. Each symbol represents an individual cell within a single histological sample from a minimum of 3 distinct samples/genotype. Error bars indicate mean ± SEM. Statistics determined using a one-way ANOVA with a Tukey post test (A, C), and denoted as follows: *p<0.05, **p<0.01, ***p<0.001.
Figure 2.
Neutrophils accumulate and have an activated phenotype in spleens of lupus-prone in the absence of BCMA.
(A) The frequency and total number of splenic neutrophils (CD11b+Ly6G+) was determined in mice of the indicated genotype at 6 and 25 weeks of age. Left panel: representative flow cytometry plots showing the percentages of neutrophils for each strain. Right panel: total numbers of neutrophils in spleens of mice were quantified from 6–9 mice/genotype. (B) Neutrophil nuclear morphology was validated using ImageStream technology by measuring co-localization of CD11b, Ly6G and the presence of a multi-lobed nucleus using DAPI. One representative image/genotype from at least 100 images/genotype is shown. Images were taken at 60X. (C) The frequency of neutrophils in spleens of Nba2 and Nba2;Tnfrsf17−/− mice were quantified from 9 mice per strain. (D) Three-month-old WT and Tnfrsf17−/− mice were injected with a single dose of either PBS or pristane. After 4 weeks, the frequency of neutrophils in spleens of mice was determined. Left panel: representative flow cytometry plots showing the percentages of neutrophils for each group. Right panel: total numbers of neutrophils for each group; each symbol represents an individual animal. Combined data from two independent experiments. (E) Representative histograms showing the activation state of neutrophils freshly isolated from spleens of mice compared to FMO (solid grey) and WT (solid black) controls. One histogram from 5 individual mice/genotype analyzed. (F) Gene expression values of Tnfrsf17 relative to HPRT in sorted cells from spleens of mice were determined by qPCR on total RNA. Combined data from three independent mice/genotype. (G) The percent of viable neutrophils was measured at the indicated time-points from purified splenic neutrophils using LIVE/DEAD Fixable AQUA. Combined data from 3 mice/genotype. Error bars indicate mean ± SEM. Statistics determined with a one-way ANOVA using a Tukey post test (A, D) or Student’s t test (C), and denoted as follows: *p<0.05, **p<0.01, ***p<0.001.
Figure 3.
Neutrophils co-localize in T cell zones and influence CD4+ T cell responses in a BAFF-dependent manner.
(A) Confocal microscopy of spleen sections demonstrating the in situ localization of neutrophils. CD11b – white, Ly6G – red, IgD – blue, PNA – green. Scale bar indicates 100 µm. (B) Confocal microscopy of spleen sections demonstrating the co-localization of neutrophils and CD4+ T cells. Ly6G – white, CD4– red, IgD – blue, PNA – green. Scale bar indicates 100 µm. Scale bar of inset indicates 20 µm. Images are representative of at least three images from 4 mice/genotype. (C) Representative histograms showing BR3 expression on CD4+ T cells from spleens of three mice/genotype. Total B cells (B220+) and a FMO stain served as controls. (D) Purified neutrophils from spleens of lupus-prone mice were co-cultured with CD4+ T cells from WT mice at a 1∶1 ratio in the presence or absence of BR3 blocking antibody. After 3 days of culture, CD4+ T cell proliferation was measured by CellTrace Violet dilution and cytokine concentrations in culture supernatant were measured by ELISA. Combined data from three mice/genotype. One of three independent experiments with similar results is shown. (E) Gene expression levels of Tnfrsf13C (BR3) in purified neutrophils from mice of the indicated genotype. Samples were normalized to HPRT and the expression in WT animals was set to one. Total B cells are shown as a positive control. Combined data from 3 mice/genotype. (F) Serum IFNγ levels from 6- and 25-week-old mice of the indicated genotype were measured by ELISA. Combined data from 5–9 mice/genotype. (G) Total splenocytes from mice of the indicated genotype were cultured in the presence or absence of PMA/ionomycin with GolgiStop. The frequency of IFNγ-producing CD4+ T cells was assessed following 5 hour in vitro stimulation. Combined data from 4 mice/genotype at 6 and 25weeks of age. Error bars indicate mean ± SEM. Statistics determined with a two-way ANOVA using a Bonferroni post test (D), or a one-way ANOVA with a Tukey post test (F, G), and denoted as follows: *p<0.05, **p<0.01, ***p<0.001, ns – not significant.
Figure 4.
Depletion of neutrophils ameliorates autoimmune disease.
B6.Faslpr/JTnfrsf17−/−mice were treated with the 1A8 neutrophil depleting antibody or a control antibody. After 4 weeks, sera, kidney and spleen were analyzed for markers of autoimmune disease. (A) Representative flow cytometry plots showing reduced frequency of neutrophils (CD11b+GR1+) in spleens after treatment. One plot from 5 mice/group is shown. (B, C) Serum BAFF and IFNγ levels were measured after treatment by ELISA. Combined data from 5 mice/genotype. (D) Total numbers of CD4+ T cells and IFNγ-producing CD4+ T cells were quantified from spleens of mice after treatment. Combined data from 5 mice/group. (E) Total numbers of B cells, GC B cells (CD19+GL7+CD138−), and PCs (CD19+/lowCD138+) in spleens of mice after treatment were determined by flow cytometry. (F) Serum dsDNA IgG titers of mice before and after treatment were determined by ELISA. Each symbol represents the fold change of autoantibody titers from an individual mouse after treatment. (G) IgG and C3 deposition in kidneys of mice was measured after treatment. One representative image from 5 mice/group is shown. (A–G) One of two independent experiments with similar results is shown. Error bars indicate mean ± SEM. Statistics determined with a Student’s t test (B–E), or a two-way ANOVA using Bonferroni post test (F), and denoted as follows: *p<0.05, **p<0.01.