Neutrophils Slow Disease Progression in Murine Lupus via Modulation of Autoreactive Germinal Centers

Abstract

Neutrophils are well characterized as mediators of peripheral tissue damage in lupus, but it remains unclear whether they influence loss of self-tolerance in the adaptive immune compartment. Lupus neutrophils produce elevated levels of factors known to fuel autoantibody production, including IL-6 and B cell survival factors, but also reactive oxygen intermediates, which can suppress lymphocyte proliferation. To assess whether neutrophils directly influence the progression of autoreactivity in secondary lymphoid organs (SLOs), we characterized the localization and cell-cell contacts of splenic neutrophils at several stages in the progression of disease in the NZB/W murine model of lupus. Neutrophils accumulate in SLO over the course of lupus progression, preferentially localizing near T lymphocytes early in disease and B cells with advanced disease. RNA sequencing reveals that the splenic neutrophil transcriptional program changes significantly over the course of disease, with neutrophil expression of anti-inflammatory mediators peaking during early-stage and midstage disease, and evidence of neutrophil activation with advanced disease. To assess whether neutrophils exert predominantly protective or deleterious effects on loss of B cell self-tolerance in vivo, we depleted neutrophils at different stages of disease. Neutrophil depletion early in lupus resulted in a striking acceleration in the onset of renal disease, SLO germinal center formation, and autoreactive plasma cell production. In contrast, neutrophil depletion with more advanced disease did not alter systemic lupus erythematosus progression. These results demonstrate a surprising temporal and context-dependent role for neutrophils in restraining autoreactive B cell activation in lupus.

Figure 1

Neutrophils accumulate in the spleen over the course of lupus progression. (A) Representative FACS plots showing frequency of Ly6G+CD11b+ neutrophils as a fraction of CD45+ splenocytes. (B) Quantitation of frequency (of CD45+Live) and numbers of Ly6G+CD11b+ splenocytes cells by FACS (n=4–5mice/group). C57BL/6 mice are 15–20-week old female mice in (A) and (B). (C) Representative IHC of spleen showing neutrophil localization relative to the marginal zone (MOMA+). 200× Mag. Analysis was done as described in Materials and Methods. n=5 mice per group. *p<0.05, **p<0.01 by unpaired Student’s t-test. Data shown are the mean ± s.e.m.

Figure 2

Neutrophil contacts with splenic B and T lymphocytes in NZB/W spleen change over the progression of lupus. (A) Representative IHC showing neutrophil proximity to B220+ cells and CD3+ cells in lupus spleen at several stages in the progression of lupus. Insets show neutrophil localization in relation to CD3+ and B220+ cells. (B) Quantitation of neutrophils in contact with B220+ B cells and CD3+ T cells as a frequency of Ly6G+ cells per 200× field. Analysis was performed as described in Materials and Methods. n=5 mice per group. *p<0.05, **p<0.01 by Mann-Whitney U-test comparing frequency of neutrophils in contact with B220+ B cells and frequency of neutrophils in contact with CD3+ T cells. Data shown are the mean ± s.e.m.

Figure 3

Transcriptional profile of splenic neutrophils is consistent with acquisition of a protective phenotype as auto-reactivity accelerates and loss of this phenotype in advanced disease. (A) Principle components analysis reveals differential gene expression (cutoff >1, norm (rlog) expression) over the course of disease. (B) Differential expression analysis was conducted for 130 select genes of immunologic interest based on neutrophil function and regulation of immune responses using deSeq. Expression heat map of normalized and rlog transformed mRNA counts quantitated via RNAseq for select genes that were differentially expressed among the 3 experimental groups (significance cutoff, FDR<0.05, ANOVA (Kruskal-Wallis). n=6/group.

Figure 4

Continuous depletion of neutrophils from 25–30 weeks of age (established disease) does not alter lupus disease progression. (A) Quantitation of the efficacy of neutrophil depletion by FACS analysis of the frequency of Gr-1hiCD11bhi splenocytes at sacrifice (30 weeks of age) following 5 weeks of treatment with anti-Ly6G (1A8) or isotype control. (B) Progression of renal disease was assessed by quantitation of proteinuria. Scoring: 0.5=trace, 1=0.3g/L, 2=1g/L, 3=3g/L, 4>20g/L. (C) Quantitation of serum anti-dsDNA IgG by ELISA at sacrifice. (D) Splenomegaly was assessed at sacrifice. (E) Splenic germinal center size & number were quantitated by immunohistochemistry as described in Materials and Methods. n=10 mice per group. Outliers > x ®±2σ were excluded from analysis in (D) and (E). ‘ns’=not significant, ***p<0.001 by Mann-Whitney U-test. Data shown are the mean ± s.e.m.

Figure 5

Continuous depletion of neutrophils from 14–26 weeks of age (disease onset) accelerates development of auto-immunity. (A) Quantitation of the efficacy of neutrophil depletion by FACS analysis of the frequency of Gr-1hiCD11bhi splenocytes at sacrifice (26 weeks of age) following 12 weeks of treatment with anti-Ly6G (1A8) or isotype control. (B) Progression of renal disease was assessed by quantitation of proteinuria. Scoring: 0.5=trace, 1=0.3g/L, 2=1g/L, 3=3g/L, 4>20g/L. (C) Serum anti-dsDNA was quantitated by ELISA at baseline (immediately before depletion began), and after 4, 6, 12 weeks of antibody treatment. (D) Splenomegaly assessed at sacrifice. Outliers > x ®±2σ were excluded from analysis in (D) n=10 mice per group. *p<0.05, **p<0.01, ***p<0.001 by Mann-Whitney U-test. Data shown are the mean ± s.e.m.

Figure 6

Germinal center formation is greatly accelerated with neutrophil depletion during the onset of autoimmunity. (A) Representative IHC of splenic germinal center formation in the isotype-treated versus anti-Ly6G-treated mice. 200× Mozaix. (B) IHC analysis was used to quantitate the extent of splenic germinal center formation. (C–D) FACS analysis was used to assess the frequency and absolute numbers of TFH cells and germinal center B cells (gating as described in Mat. & Met.). N=10 mice per group (B–D). Outliers > x+2d were excluded from analysis in (C) and (D). *p<0.05, **p<0.01, ***p<0.001 by Mann-Whitney U-test. Data shown are the mean + s.e.m.

Figure 7

Changes in frequency of anti-dsDNA+ and IgG+ antibody secreting cells (ASC) in spleen, kidney, and bone marrow. ELISPOT quantitation of (A) absolute number of IgG+ ASC per whole tissue, (B) absolute number of anti-dsDNA+IgG+ ASC per whole tissue, (C) frequency of IgG+ ASC per 106 cells, (D) frequency of anti-dsDNA+IgG+ ASC per 106 cells. (E) Anti-dsDNA IgG-secreting cells as a fraction of the total IgG-secreting cells in bone marrow. n=10 mice per group. Outliers > x ®±2σ were excluded from analysis. Zero value data points are not shown on graphs due to log scale but were included in the analysis. *p<0.05, **p<0.01, ***p<0.001 by Mann-Whitney U-test. Data shown are the mean ± s.e.m.