Neutrophils accumulate and have an activated phenotype in spleens of lupus-prone in the absence of BCMA.

<p>(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 <i>Nba2</i> and <i>Nba2;Tnfrsf17^−/−</i> mice were quantified from 9 mice per strain. (D) Three-month-old WT and <i>Tnfrsf17^−/−</i> 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 <i>Tnfrsf17</i> relative to <i>HPRT</i> 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.</p

Neutrophils co-localize in T cell zones and influence CD4⁺ T cell responses in a BAFF-dependent manner.

<p>(A) Confocal microscopy of spleen sections demonstrating the <i>in </i><i>situ</i> 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 <i>Tnfrsf13C</i> (BR3) in purified neutrophils from mice of the indicated genotype. Samples were normalized to <i>HPRT</i> 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 <i>in </i><i>vitro</i> 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.</p

Neutrophils and dendritic cells contribute to elevated BAFF levels in the absence of BCMA.

<p>(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.</p

Inflammatory metabolites are increased in the MCT-treated rat lung.

<p>Inflammation, via INF-gamma or TNF-alpha, activates indoleamine-2,3-dioxygenase (IDO) or tryptophan-2,3-dioxygenase (TDO) that degrade tryptophan to kynurenine/kynurenate (center pathway). There is a significant increase in tryptophan, kynurenine, kynurenate and serotonin in the pre-PH lung suggestive of increased inflammation. (N = 10, p<0.05).</p

A shift in the balance omega 6 and omega 3 fatty acids and increased pro-inflammatory eicosanoid production in the MCT-treated rat lung.

<p>The ratio between pre-PH and control metabolites show both significant increases (red boxes) for omega 6, omega 3 fatty acids and eicosanoids and a trending decrease (light green box) for stearidonate (A). The pro-inflammatory prostaglandins E2, D2, J2 and leukotriene B5 are also significantly increased in the pre-PH lung (N = 10, p<0.05, B).</p

Evidence of a redox imbalance in the MCT-treated rat lung.

<p>Despite an increase in glutathione recycling, as indicated by increases in the 5-oxoproline metabolite, both reduced and oxidized glutathione are significantly decreased in the pre-PH (A), indicative of increased oxidative stress. The ratio between pre-PH and control metabolites demonstrate significant increases (red boxes) gamma-glutamyl amino acids (B). Activation of the gamma-glutamyl cycle is usually associated with an increased inflammatory response (N = 10, p<0.05).</p

Disruption of arginine metabolism in the MCT-treated rat lung.

<p>The metabolic fate of arginine is complex being involved in NO signaling, the urea cycle, proliferation, and matrix remodeling (center pathway diagram). There is significantly increased urea production in the pre-PH lung indicative of increased arginase activity. Aspartate and fumarate, which are involved in arginine biosynthesis, are significantly increased in the pre-PH lung as is arginine itself. Polyamine metabolites are significantly increased in the pre-PH lung, indicative of increased cellular proliferation while the significant increase in proline pathway metabolites is suggestive of extracellular matrix remodeling (N = 10, p<0.05).</p

Carnitine homeostasis is altered in the MCT-treated rat lung.

<p>Significant decreases in conjugated acyl carnitines such as palmitoylcarnitine, stearoylcarnitine and oleoylcarnitine indicate that there is disrupted fatty acid transport to mitochondria in the lungs of pre-PH rats (N = 10, p<0.5).</p

Biomarkers of cellular damage are increased in the MCT-treated rat lung.

<p>Phospholipid degradation and membrane remodeling markers (ethanolamine and glycerophosphoethanolamine) are significantly increased in the pre-PH lung. Methylhistidines (1-methylhhistidine and N-acetyl-3-histidine), produced by methylation of actin and myosin in muscle, are indicative of muscle protein breakdown and therefore muscular damage. Significant increases in mitochondrial membrane degradation (2-stearoylglycerophosphoglycerol) and the breakdown product of mitochondrially-encoded/synthesized proteins (N-formylmethionine) reflect mitochondrial damage. (N = 10, p<0.05).</p

Evidence for a glycolytic switch in the MCT-treated rat lung.

<p>Data for control lung are represented in grey boxes and data for the pre-PH lung are represented in blue boxes. Quantities are in arbitrary units (N = 10, p<0.05). MCT-treated animals have higher levels of glucose, glucose-6-phosphate, glucose-1-phosphate, fructose, fructose-6-phosphate, 6-phospho gluconate, ribulose 5-phosphate, sedoheptulose-7-phosphate, pyruvate and lactate. These metabolic data are consistent with an upregulation of glycolytic or glucose dependent pathways in the lungs of pre-PH rats.</p