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.

This article is featured in In This Issue, p.379

A central feature of lupus pathogenesis is production of autoantibodies that target multiple organ systems, resulting in chronic inflammation and, in severe cases, life-threatening organ damage. However, the mechanisms underlying the dysregulated adaptive immune response and progression of autoreactivity in lupus remain incompletely characterized. Although B and T cells are well-known to play a key role in systemic lupus erythematosus (SLE) disease pathogenesis (1), the contribution of innate immune cells to disease is increasingly recognized (2). Neutrophils help orchestrate the outcome of immune responses by tailoring their effector program to an acute inflammatory response during infection, immunosuppression during chronic inflammatory states such as malignancy, or resolution of inflammation to promote healing. In SLE, neutrophils are known to mediate tissue damage and act as a source of self-nucleic acids that drive aberrant plasmacytoid dendritic cell activation (35). Neutrophil-specific components are targeted by autoantibodies in 10–20% of SLE patients, likely because of a high neutrophil turnover rate, impaired dying cell clearance, and elevated propensity for neutrophil extracellular trap formation (NETosis) (411). Because NETosis involves extravasation of nucleic acid and histone, this form of cell death may provide antigenic fuel for autoreactive lymphocytes, as well as IFN-producing plasmacytoid dendritic cells (3, 4). In vivo inhibition of NETosis with peptidylarginine deiminase 4 inhibitors in murine lupus models ameliorates vascular, dermal, and renal tissue pathology and reduces systemic IFN-I signaling (10, 12).

In contrast, recent literature has contended that neutrophils in lupus, similar to neutrophils in other inflammatory conditions such as malignancy and sepsis, may acquire an immunosuppressive phenotype and restrain development of autoreactivity (13, 14). In an amyloid-induced model of systemic autoimmunity, neutrophil production of reactive oxygen species (ROS) mediates suppression of systemic IFN-γ levels and autoantibody production (15). In addition, in vitro coculture of splenic neutrophils with B lymphocytes isolated from the NZB/W lupus model demonstrated that neutrophils can suppress B cell Ig production. Interestingly, neutrophil capacity for suppression of B cells was only apparent early in disease, suggesting that the neutrophil effector program may change with disease progression (16). Neutrophils may have different effects on adaptive immunity depending on the type of cell encounter. Thus, splenic neutrophils isolated in murine lupus were also able to suppress regulatory T cell (Treg) differentiation in vitro, effects that should propagate autoimmunity (1618). However, little is known about which immune cell types neutrophils interact with in vivo. It has been suggested that the predominant effect of neutrophil ROS in SLE may be protective, as was demonstrated by the finding of accelerated autoimmunity in MRL/lpr mice deficient in NADPH oxidase (NOX2, the enzyme responsible for oxidative burst in neutrophils) (19). Aside from ROS, circulating neutrophils in SLE also produce elevated amounts of proinflammatory mediators including IL-6, TNF-α, B cell activation factor (BAFF), and a proliferation-inducing ligand (APRIL), factors known to promote adaptive immune dysregulation in lupus (4, 2022). The cytokine profile of neutrophils in SLE remains incompletely characterized, particularly neutrophil production of anti-inflammatory factors such as TGF-β and IL-1RA.

Thus, although recent literature highlights that neutrophils influence the course of autoimmune disease, a more complete in vivo model is needed to reconcile the conflicting data pointing toward both pathogenic and regulatory effects on the adaptive immune compartment. We hypothesize in this article that neutrophils have the capacity for either deleterious or protective influence on the development of autoreactivity, dependent on variables including stage and severity of disease, tissue localization, cellular interactions, and differences in inflammatory pathways driving SLE. In this article, we assess whether neutrophils contribute directly to loss of B cell self-tolerance within secondary lymphoid tissues using the NZB/W murine model of lupus. Specifically, we characterize the localization, changing cellular contacts, and transcriptional program of splenic neutrophils at several stages during the course of autoimmunity. We find that neutrophils are in close proximity to splenic T and B lymphocytes, and that lymphocyte–neutrophil contacts in the spleen change over the course of disease. Transcriptome profiling of splenic neutrophils demonstrates that neutrophils display a regulatory transcriptional program that is apparent during the initiation of disease but is lost with advanced disease. Consistent with this result, neutrophil depletion early in disease resulted in a striking acceleration in the development of B cell autoreactivity and renal proteinuria, a result that was not apparent in advanced disease. Collectively, our findings demonstrate that the neutrophil effector program and the nature of neutrophil impact on autoimmunity are strongly dependent on the stage and severity of disease, a critical consideration in the assessment of neutrophils as a potential therapeutic target in lupus.

Female (NZB×NZW)F1 mice were purchased from The Jackson Laboratory and housed in the pathogen-free animal facility at the University of Rochester. All murine experiments were conducted in accordance with the policies established by the University of Rochester’s University Committee of Animal Resources and the Institutional Animal Care and Use Committee.

Female (NZB×NZW)F1 mice were injected i.p. with neutrophil-depleting Ab anti-Ly6G (clone 1A8; BioXCell) or ChromPure rat whole IgG isotype control (Jackson ImmunoResearch) using 500 μg of Ab per mouse every 2 d over the entirety of the indicated depletion periods. Mice were sacrificed 24 h after the last Ab injection. Flow cytometry was used to verify the efficacy of neutrophil depletion.

Proteinuria was quantitated using urinalysis reagent sticks (Teco Diagnostics). At sacrifice, kidneys were formalin fixed for histological analysis. Kidney sections (4 μm) were stained with H&E. Pathology was analyzed and scored in a blinded fashion (B.I. Goldman) (23). In brief, the severity of glomerular, interstitial, and vascular lesions was determined on a scale of 0 to 4+. Multiple sections at a minimum of two different levels were examined, with each section typically containing >50 glomeruli and >25 blood vessels. Blood was collected by submandibular bleed, and serum anti-dsDNA IgG concentration was determined by ELISA (Alpha Diagnostic International).

Bone marrow was isolated by flushing of murine femur and tibia with RPMI 1640. Splenocytes were isolated by mechanical disaggregation. Erythrocytes were lysed with ammonium chloride buffer and leukocytes stained for FACS analysis with an LSR II (Becton-Dickinson). Lymphocyte populations were identified as CD3+CD4+CD8 (CD4+ T cells), CD3+CD4+CD8CD25+Foxp3+ (Tregs), CD3+CD4+CD8CXCR5hiPD1+ICOS+ (T follicular helper [Tfh] cells), CD3CD19+ (B cells), and CD19+CD95+Peanut-agglutinin+ (germinal center [GC] B cells). Myeloid cells were identified using B220+/CD4+/CD8+ cell exclusion and expression of CD11b+Gr-1hiLy6Cint (neutrophils) and CD11b+Gr-1lo-intCD11cF4/80hi (monocytes/macrophages), which were further subgated into Ly6CloCX3CR1hiSSClo (M2 macrophage) or Ly6ChiCX3CR1loSSClo (M1 macrophage). Dead cells were excluded from all analyses using AQUA (Invitrogen). Abs used for flow cytometry were anti-CD3 (145-2C11), anti-CD4 (GK1.5), anti-CD8 (53-8.7), anti-CXCR5 (L138D7), anti-Foxp3 (150D), anti-CD25 (3C7), anti-PD1 (29F.1A12), anti-ICOS (15F9), anti-CD19 (6D5), anti-CD95 (SA367H8), anti-B220 (RA3-6B2), anti–Gr-1 (RB6-8C5), anti-Ly6C (HK1.4), and anti-CX3CR1 (SA011F11).

Freshly isolated spleen was snap-frozen in OCT buffer and cut into 4-μm sections for immunohistochemical staining. Sections were blocked (5% normal donkey serum/PBS), Ab-labeled, and mounted in Prolong Gold Antifade with DAPI (Life Technologies). Tissue sections were imaged with Zeiss Axioplan Microscope at 200× magnification for all images. AxioVision software (Zeiss) was used for morphometric analyses. GCs were identified as PNA+PCNA+B220dim cell clusters and follicles as clusters of B220bright cells. The marginal zone was identified as a line of MOMA+ staining in the peripheral region B220+ follicles. The extent of GC formation was calculated by dividing the total area occupied by GC by the total area of the tissue section. Neutrophil contacts with B and T lymphocytes in the spleen were quantitated based on the number of neutrophils (Ly6Gbright cells with neutrophil morphology) that appeared to be in contact with B220+ or CD3+ cells in the white pulp. Neutrophil localization relative to primary and secondary splenic follicles, as well as to the marginal zone (MOMA+ line), was quantitated by positioning splenic follicles in a consistent fashion within the 200× field and measuring the distance between Ly6G+ cells and the follicle edge (B220+ cell clusters) or MOMA+ marginal zone line. Stains used for immunohistochemistry (IHC) were anti-B220 (RA3-6B2), peanut agglutinin, anti-PCNA (PC10), anti-MOMA (MOMA-2), anti-Ly6G (1A8), and anti-CD3 (145-2C11).

ELISPOT was performed as described previously (23, 24). In brief, to detect anti-dsDNA–secreting cells, we coated 96-well MultiScreen plates (Millipore) with poly-l-lysine and incubated with calf thymus DNA. Plates were coated with goat anti-mouse IgG (Fc fragment specific; Jackson ImmunoResearch Laboratories) to detect total IgG-secreting cells. Cell suspensions from spleen, bone marrow, and kidney were added to individual wells, starting with 5 × 105 cells in the top row and preforming a 2-fold serial dilution. Cells were incubated overnight at 37°C and 5% CO2. After incubation, plates were washed several times with 0.1% Tween 20 in PBS, incubated with alkaline phosphatase-goat anti-mouse IgG Ab (Southern Biotech), and finally developed with Vector Blue alkaline phosphatase substrate III kit (Vector Laboratories). Ab-secreting cells (ASCs) were enumerated with a stereoscopic microscope (Cellar Technology).

Splenic neutrophils were purified by mechanical disaggregation of freshly isolated spleen without the use of erythrocyte lysis. Splenic neutrophils were isolated for RNA sequencing analysis pre-enrichment by magnetic negative selection (Miltenyi), followed by sorting of CD11bhiLy6GhiLy6bhiLy6Cint cells with doublet and dead (DAPI+) cell exclusion to obtain a purity of ≥97% CD11bhiLy6Ghi cells, as determined by postsort analysis. Neutrophils were kept cold (at 4°C) throughout the entire course of neutrophil isolation and handled gently to avoid mechanical activation. Abs used for FACS purification were anti-Ly6B (REA115), anti-Ly6C (HK1.4), anti-Ly6G (1A8), and anti-CD11b (M1/70).

Neutrophil total RNA was isolated using the RNeasy Plus Micro Kit (Qiagen), and RNA quality was assessed with the Agilent Bioanalyzer 2100 (Agilent) (n = 6 mice per three age groups). One nanogram of total RNA was preamplified with the SMART-Seq v4 Ultra Low Input RNA Kit for Sequencing (Takara Bio). The quantity and quality of the subsequent cDNA was determined using the Qubit Fluorometer (Thermo Fisher Scientific) and the Agilent Bioanalyzer. One nanogram of cDNA was used to generate Illumina-compatible sequencing libraries with the NexteraXT (Illumina) library preparation kit. The amplified libraries were hybridized to the Illumina single end flow cell and amplified using the cBot (Illumina) at a concentration of 10 pM per lane. Single-end reads of 100 nt were generated for each sample and aligned to the organism-specific reference genome. Sequenced reads were cleaned using Trimmomatic-0.32 before mapping to the mouse reference genome (GRCm38.p4) with STAR-2.4.2a. Raw read counts were obtained using HTSeq and gencode M6 mouse gene annotations. DESeq2 1.12.3 was used to perform data normalization. After an expression level cutoff of 1 (rlog DESeq norm counts), principle components analysis was performed using all transcripts. In addition, a list of 130 genes of immunologic interest was compiled and differential analysis was conducted using DESeq. These include genes involved in neutrophil regulation of immune responses, both protective mediators and proinflammatory mediators, as well as genes involved in neutrophil responses to inflammation such as IFN-regulated genes and cytokine signaling genes like the Stat family. The full transcriptome data set is available at the Gene Expression Omnibus (accession number GSE97439; https://www.ncbi.nlm.nih.gov/geo/).

Statistical analysis comparing experimental groups was done using the Mann–Whitney U test or Kruskal–Wallis ANOVA using GraphPad Prism and MultiExperiment Viewer software. Differential expression analyses of RNA sequencing data were conducted using a Benjamini–Hochberg correction (false discovery rate [FDR] ≤ 0.05). Principal components analysis was conducted using JMP Pro 13. Significance levels are as follows: *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

Under homeostatic conditions, neutrophils are present in secondary lymphoid organ (SLO) tissues at a frequency of 1–2% of total splenocytes, although their numbers can increase dramatically during an immune response (25). To characterize neutrophils in lupus and their impact on adaptive immunity, we first quantitated neutrophil infiltration of the spleen at several points over the course of lupus progression by FACS analysis of splenocytes from female NZB/W mice at 11, 23, 25, and 30 wk of age. Between 23 wk of age (around the onset of overt pathology, including proteinuria) and 30 wk of age (advanced lupus), we observed a significant increase in both the frequency and the total numbers of neutrophils infiltrating the spleen (Fig. 1A, 1B). Neutrophils were observed predominantly in the red pulp and interfollicular areas of the white pulp (Fig. 1C), and although we observe infiltration with the progression of disease, the average neutrophil proximity to B cell follicles did not change over the course of disease, nor were they detected in GCs (data not shown).

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 = 4–5 mice per group). C57BL/6 mice are 15- to 20-wk-old female mice in (A) and (B). (C) Representative IHC of spleen showing neutrophil localization relative to the marginal zone (MOMA+). Original magnification ×200. Analysis was done as described in the 2Materials and Methods. n = 5 mice per group. Data shown are the mean ± SEM. **p < 0.01 by unpaired Student t test.

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 = 4–5 mice per group). C57BL/6 mice are 15- to 20-wk-old female mice in (A) and (B). (C) Representative IHC of spleen showing neutrophil localization relative to the marginal zone (MOMA+). Original magnification ×200. Analysis was done as described in the 2Materials and Methods. n = 5 mice per group. Data shown are the mean ± SEM. **p < 0.01 by unpaired Student t test.

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To address the hypothesis that neutrophils are directly interacting with splenic B and T lymphocytes, we quantified neutrophil contacts with B and T cells by IHC of spleens isolated from 11-, 20-, and 29-wk-old NZB/W mice. Neutrophils were observed in close contact with T and B lymphocytes in the splenic white pulp during all stages of disease (Fig. 2A). At 11 wk, early in disease, the frequency of neutrophil contacts with T cells was 1.5-fold higher than contacts with B cells (Fig. 2B). By 29 wk of age, this phenomenon is reversed, with nearly half of all Ly6G+ neutrophils in the white pulp in close contact with B220+ B cells, and only 20% of neutrophils in contact with CD3+ T cells. The changing frequency of neutrophil contacts with splenic lymphocytes over the course of disease suggests that the functional impact neutrophils have on splenic lymphocyte populations changes over the course of disease.

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+ 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, with white arrows denoting close cell contacts. (B) Quantitation of neutrophils in contact with B220+ B cells and CD3+ T cells as a frequency of Ly6G+ cells (original magnification ×200) field. Analysis was performed as described in the 2Materials and Methods. n = 5 mice per group. Data shown are the mean ± SEM. *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.

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+ 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, with white arrows denoting close cell contacts. (B) Quantitation of neutrophils in contact with B220+ B cells and CD3+ T cells as a frequency of Ly6G+ cells (original magnification ×200) field. Analysis was performed as described in the 2Materials and Methods. n = 5 mice per group. Data shown are the mean ± SEM. *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.

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Because we observed significant changes in neutrophil frequency and localization relative to splenic B and T leukocytes in the spleen over the course of disease, we hypothesized that neutrophil function changes with disease progression. Because very little is known about the neutrophil effector program within the SLO in autoimmunity, we used RNA sequencing as an unbiased approach to assess whether splenic neutrophils display a regulatory or proinflammatory transcriptional program, as well as to determine whether this program changes over the course of disease. Neutrophils were pre-enriched and FACS sorted from NZB/W splenocytes at three time points: 14 wk (early disease), 20 wk (middisease), and 26 wk (advanced disease) (26). Neutrophil mRNA was isolated and the transcriptome sequenced using an Illumina platform.

Principal components analysis of the entire neutrophil transcriptome revealed clear separation of neutrophils isolated at the three disease time points, indicating significant changes in the neutrophil transcriptional program with lupus progression (Fig. 3A). Differential expression analysis (using a significance cutoff of FDR < 0.05) revealed several transcripts of immunologic interest (Fig. 3B), including TNF-α (Tnf) and BAFF (Tnfsf13b), factors known to promote B cell autoreactivity in SLE (2729). Interestingly, these two factors show a relative decrease at 20 wk of age, followed by an increase with more advanced disease (26 wk) (Fig. 3B). Concomitantly, between 14 and 20 wk, we observed an increase in proresolving and anti-inflammatory mediators including PD-L1 (Cd274), IL-1RA (Il1rn), and TGF-β (Tgfb1) (Fig. 3B). These results suggest that, during the onset and acceleration of autoantibody production in NZB/W mice between 14 and 20 wk of age, splenic neutrophils adopt a more regulatory transcriptional program.

FIGURE 3.

Transcriptional profile of splenic neutrophils is consistent with acquisition of a protective phenotype as autoreactivity 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 RNA sequencing for select genes that were differentially expressed among the three experimental groups (significance cutoff, FDR < 0.05, ANOVA [Kruskal–Wallis]). n = 6 per group.

FIGURE 3.

Transcriptional profile of splenic neutrophils is consistent with acquisition of a protective phenotype as autoreactivity 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 RNA sequencing for select genes that were differentially expressed among the three experimental groups (significance cutoff, FDR < 0.05, ANOVA [Kruskal–Wallis]). n = 6 per group.

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The increase in protective, anti-inflammatory mediators by 20 wk coincides with an increase in Stat3 and Socs3, transcription factors involved in induction of an anti-inflammatory neutrophil phenotype in response to prolonged exposure to an inflammatory cytokine milieu (30, 31). It is notable that expression of genes associated with restraining inflammation, including PD-L1 (Cd274), IL-1RA (Il1rn), and TGF-β (Tgfb1), is downregulated between 20 and 26 wk. Concomitantly, genes involved in neutrophil responses to inflammation and activation increased during this time frame. This includes the alarmins S100a8 and S100a9 (32), which have largely, although not exclusively, been associated with proinflammatory functions (33, 34). The IFN-regulated chemokines, CXCL9 (MIG), CXCL10 (IP-10), and CXCL11 (I-TAC), important for T cell chemoattraction and activation were also significantly upregulated (35) at 26 wk (Fig. 3B). Based on these findings, we hypothesize that neutrophils acquire a protective transcriptional program between 14 and 20 wk of age during the initial rise in autoreactivity in NZB/W mice, but that the neutrophil phenotype shifts to a more activated state by 26 wk (advanced disease).

To examine whether neutrophils exert predominantly protective or deleterious effects in modulation of autoreactivity in lupus, we depleted neutrophils continuously in female NZB/W mice using an anti-Ly6G (1A8) Ab or isotype control (polyclonal rat IgG) over a period from 25 to 30 wk of age. Anti-Ly6G injection effectively depleted neutrophils, as quantitated by FACS analysis of splenocytes isolated at sacrifice (30 wk) (Fig. 4A). However, neutrophil depletion from 25 to 30 wk of age did not result in a significant alteration in the progression of disease, including proteinuria, serum anti-dsDNA levels, splenomegaly, or GC formation (Fig. 4B–E). These results suggest that neutrophils are not required for progression of autoimmunity once active disease is well established.

FIGURE 4.

Continuous depletion of neutrophils from 25 to 30 wk 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 wk of age) after 5 wk 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 = 0.3 g/l, 2 = 1 g/l, 3 = 3 g/l, 4 > 20 g/l. (C) Quantitation of serum anti-dsDNA IgG by ELISA at sacrifice. (D) Splenomegaly was assessed at sacrifice. (E) Splenic GC size and number were quantitated by IHC as described in the 2Materials and Methods. n = 10 mice per group. Values outside ± 2σ were excluded from analysis in (D) and (E). Data shown are the mean ± SEM. ***p < 0.001 by Mann–Whitney U test. ns, not significant.

FIGURE 4.

Continuous depletion of neutrophils from 25 to 30 wk 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 wk of age) after 5 wk 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 = 0.3 g/l, 2 = 1 g/l, 3 = 3 g/l, 4 > 20 g/l. (C) Quantitation of serum anti-dsDNA IgG by ELISA at sacrifice. (D) Splenomegaly was assessed at sacrifice. (E) Splenic GC size and number were quantitated by IHC as described in the 2Materials and Methods. n = 10 mice per group. Values outside ± 2σ were excluded from analysis in (D) and (E). Data shown are the mean ± SEM. ***p < 0.001 by Mann–Whitney U test. ns, not significant.

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Although neutrophil depletion during established disease did not significantly alter the course, we next assessed whether neutrophils modulate adaptive immune responses at early stages of lupus. We depleted neutrophils as described earlier, continuously in NZB/W mice over a 12-wk period, from 14 to 26 wk of age. The efficacy of depletion was assessed by FACS analysis of splenocytes (Fig. 5A), peripheral blood, and bone marrow (Supplemental Fig. 1A, 1B) isolated at sacrifice. After anti-Ly6G treatment, mice exhibited an early and significant increase in plasma anti-dsDNA levels compared with isotype-treated mice and developed proteinuria at an accelerated rate (Fig. 5B, 5C). Comparison of kidney histopathology did not reveal a statistically significant difference between anti-Ly6G– and isotype-treated groups (data not shown). Depletion during this 12-wk period also resulted in significantly increased splenomegaly (Fig. 5D) and enhanced GC formation (Fig. 6A, 6B).

FIGURE 5.

Continuous depletion of neutrophils from 14 to 26 wk of age (disease onset) accelerates development of autoimmunity. (A) Quantitation of the efficacy of neutrophil depletion by FACS analysis of the frequency of Gr-1hiCD11bhi splenocytes at sacrifice (26 wk of age) after 12 wk 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 = 0.3 g/l, 2 = 1 g/l, 3 = 3 g/l, 4 > 20 g/l. (C) Serum anti-dsDNA was quantitated by ELISA at baseline (immediately before depletion began) and after 4, 6, and 12 wk of Ab treatment. (D) Splenomegaly was assessed at sacrifice. Values outside ± 2σ were excluded from analysis in (D). n = 10 mice per group. Data shown are the mean ± SEM. *p < 0.05, ***p < 0.001, ****p < 0.0001 by Mann–Whitney U test.

FIGURE 5.

Continuous depletion of neutrophils from 14 to 26 wk of age (disease onset) accelerates development of autoimmunity. (A) Quantitation of the efficacy of neutrophil depletion by FACS analysis of the frequency of Gr-1hiCD11bhi splenocytes at sacrifice (26 wk of age) after 12 wk 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 = 0.3 g/l, 2 = 1 g/l, 3 = 3 g/l, 4 > 20 g/l. (C) Serum anti-dsDNA was quantitated by ELISA at baseline (immediately before depletion began) and after 4, 6, and 12 wk of Ab treatment. (D) Splenomegaly was assessed at sacrifice. Values outside ± 2σ were excluded from analysis in (D). n = 10 mice per group. Data shown are the mean ± SEM. *p < 0.05, ***p < 0.001, ****p < 0.0001 by Mann–Whitney U test.

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FIGURE 6.

GC formation is greatly accelerated with neutrophil depletion during the onset of autoimmunity. (A) Representative IHC of splenic GC formation in the isotype-treated versus anti-Ly6G–treated mice. Mozaix (original magnification ×200). (B) IHC analysis was used to quantitate the extent of splenic GC formation. (C and D) FACS analysis was used to assess the frequency and absolute numbers of Tfh cells and GC B cells (gating as described in 2Materials and Methods). n = 10 mice per group (B–D). Values outside x ± 2δ were excluded from analysis in (C) and (D). Data shown are the mean + SEM. *p < 0.05, ***p < 0.001 by Mann–Whitney U test. ns, not significant.

FIGURE 6.

GC formation is greatly accelerated with neutrophil depletion during the onset of autoimmunity. (A) Representative IHC of splenic GC formation in the isotype-treated versus anti-Ly6G–treated mice. Mozaix (original magnification ×200). (B) IHC analysis was used to quantitate the extent of splenic GC formation. (C and D) FACS analysis was used to assess the frequency and absolute numbers of Tfh cells and GC B cells (gating as described in 2Materials and Methods). n = 10 mice per group (B–D). Values outside x ± 2δ were excluded from analysis in (C) and (D). Data shown are the mean + SEM. *p < 0.05, ***p < 0.001 by Mann–Whitney U test. ns, not significant.

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To assess whether neutrophil depletion alters the frequency of other immune cells, we used FACS analysis to quantitate the frequency and absolute numbers of M1 and M2 macrophages, GC B cells, Tfh cells, and Foxp3+ Tregs. Consistent with the finding that GC formation is significantly augmented with anti-Ly6G treatment, we found a significant increase in numbers of GC B cells and frequency of Tfh cells after neutrophil depletion. The increase in Tfh cell frequency was not a global effect on T cells because there was no significant change in Foxp3+ Treg frequencies or absolute numbers in the spleen (Supplemental Fig. 2A). We also did not observe significant alterations in frequency of monocyte/macrophage populations or a shift in polarization to an M1 versus M2 phenotype after neutrophil depletion (Supplemental Fig. 2B, 2C). Together these results suggest that neutrophil depletion during the onset of lupus results in acceleration in the progression of autoreactivity and magnitude of the adaptive immune dysregulation.

Because the accelerated rise in serum anti-dsDNA levels seen with neutrophil depletion (Fig. 5C) may be attributable to either Ab production or Ab clearance (including precipitation of immune complexes), we quantitated the production of both total IgG and anti-dsDNA IgG ASCs directly by ELISPOT. Notably, total IgG and anti-dsDNA ASCs increase significantly in spleen, kidney, and bone marrow after anti-Ly6G treatment (Fig. 7A–D). In addition, there was enrichment in autoreactive anti-dsDNA IgG ASCs as a fraction of total IgG ASCs in the bone marrow (Fig. 7E), suggesting that neutrophil depletion beginning at the onset of autoimmunity results in a systemic increase in autoreactive B cells. Overall, these results indicate that neutrophils have a protective function during the early stages of lupus, but that this protective effect is lost by advanced stages of disease.

FIGURE 7.

Changes in frequency of anti-dsDNA+ and IgG+ ASCs in spleen, kidney, and bone marrow. ELISPOT quantitation of (A) absolute number of IgG+ ASCs per whole tissue, (B) absolute number of anti-dsDNA+IgG+ ASCs per whole tissue, (C) frequency of IgG+ ASCs per 106 cells, and (D) frequency of anti-dsDNA+IgG+ ASCs 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. Values outside ± 2σ were excluded from analysis. Zero value data points are not shown on graphs because of log scale but were included in the analysis. Data shown are the mean ± SEM. *p < 0.05, **p < 0.01, by Mann–Whitney U test. ns, not significant.

FIGURE 7.

Changes in frequency of anti-dsDNA+ and IgG+ ASCs in spleen, kidney, and bone marrow. ELISPOT quantitation of (A) absolute number of IgG+ ASCs per whole tissue, (B) absolute number of anti-dsDNA+IgG+ ASCs per whole tissue, (C) frequency of IgG+ ASCs per 106 cells, and (D) frequency of anti-dsDNA+IgG+ ASCs 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. Values outside ± 2σ were excluded from analysis. Zero value data points are not shown on graphs because of log scale but were included in the analysis. Data shown are the mean ± SEM. *p < 0.05, **p < 0.01, by Mann–Whitney U test. ns, not significant.

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Neutrophils play a key role in the innate immune response to infections, but they have increasingly been recognized to contribute to autoimmunity in diverse ways. Somewhat unexpectedly given the dominance of literature (mostly in vitro) supporting a proinflammatory role for neutrophils in lupus (2, 4, 8), we find that depletion of neutrophils early in murine SLE profoundly accelerates disease. This was evidenced by significant enhancement of splenic GC reactions and generation of autoantibody-secreting cells in SLO. Notably, autoantibody-secreting cells accumulated in target tissue (kidney), as well as the bone marrow, suggesting systemic effects of neutrophil absence on the autoimmune process. The findings that late neutrophil depletion had no impact on disease, shift in splenic neutrophil interactions from T cells to B cells, and change in transcriptional profile over the course of the disease highlight the plasticity in neutrophil roles in SLE. Our results indicate a dominantly suppressive effect for much of the disease likely mediated by direct interaction with T and B cells, whereas neutrophils acquire a more activated phenotype later in disease.

The finding that B cell autoimmunity is profoundly accelerated by neutrophil depletion starting early in disease strongly implicates a direct role for neutrophils in modulating the adaptive immune response in SLE. Although neutrophils have been shown to act in an immunosuppressive capacity during chronic inflammatory conditions such as malignancy or viral infection, the possibility that a proresolving or protective neutrophil is present in lupus has only recently been proposed (15, 16, 3639). In an amyloid-induced lupus model, neutrophil depletion worsened disease similar to our findings. A regulatory effect for neutrophils was suggested to be mediated by ROS inhibition of NK cell IFN-γ production at local sites of inflammation (15). Another study demonstrated that Gr-1hiCD11b+ splenocytes suppress lupus pathogenesis in young male, but not female, NZB/W mice based on the finding that administration of anti–Gr-1 Ab in vivo increased anti-dsDNA Ab titers (16). Limitations of this study include the use of anti–Gr-1 Ab, which depletes both neutrophils and some monocyte populations (4042). To our knowledge, ours is the first study in a well-accepted murine lupus model demonstrating that specific (anti-Ly6G) depletion of neutrophils accelerates disease via promotion of GC reactions.

Nonetheless, this finding should be considered in the context of literature implicating neutrophils as exerting a highly pathogenic impact on loss of tolerance in certain contexts. Neutrophils in both peripheral blood and bone marrow have been shown to express elevated amounts of BAFF, a factor known to underlie failure of autoreactive B cell anergy (20, 27, 43, 44). Along these lines and in contrast with our findings, neutrophil depletion in a B6.Faslpr/J/Tnfrsf17−/− autoimmune model resulted in significant amelioration of autoimmunity, including reduced serum anti-dsDNA titers and splenic plasma cell frequency. This result depended on splenic neutrophil production of BAFF, which was shown in vitro to stimulate CD4+ T cell production of IFN-γ, a critical mediator of autoreactive GC formation (21, 44). The contrasting results in this model may be attributable to the strong dependence of the lupus phenotype on specific immune pathways, such as BAFF signaling. We do not rule out the possibility that neutrophils can augment the progression of autoreactivity in the NZB/W model, particularly during advanced disease, where we observed a notable rise in transcription of BAFF and TNF-α between 20 and 26 wk. It is also possible that other neutrophil functions in addition to proinflammatory cytokine secretion, such as NETosis, are dominantly proinflammatory. On the other hand, it is interesting to note that when NETs were inhibited in the NZM lupus model, vascular damage was ameliorated, but anti-DNA Abs actually did increase (10, 12), suggesting that even NETosis may have unexpected anti-inflammatory effects (45). It is also important to consider that the lack of effect of neutrophil depletion from 25 to 30 wk of age in our experiments may be attributable to neutrophil effects being overwhelmed by a strong immune response and rapidly worsening autoimmunity in this period. Nonetheless, our findings clearly demonstrate that neutrophils are nonessential for progression of both autoreactivity and tissue pathology during this time frame, regardless of the precise mechanisms of contribution to the disease.

Our analysis of neutrophil contacts in the spleen reveals that neutrophils are preferentially localized near T cells early in disease, with a shift toward predominant B cell interactions with advanced disease. These data, in conjunction with the results of neutrophil depletion, suggest that neutrophils play a protective role via interaction with splenic T cells. Prior studies have investigated the effects of splenic neutrophils on lymphocyte proliferation and effector function in vitro but with apparently conflicting results. Thus, Trigunaite et al. (16) reported that NZB/W splenic Gr-1highCD11b+ cells suppressed B cell differentiation to ASCs in vitro, suggesting that splenic neutrophils play a protective role and that this is consistent with worsening disease upon in vivo anti–Gr-1 Ab administration in male mice. Interesting differences were noted in the mechanisms of suppression by neutrophils isolated from young male versus female mice, with an ROS and NO dependence only in neutrophils from female mice. Similar to our study, a plasticity in neutrophil function was suggested by the loss of the suppressive phenotype in neutrophils from older mice (16). This same group also described Gr-1+ cell inhibition of Tfh cell differentiation in vitro and correspondingly increased Tfh cells with in vivo depletion of Gr-1+ cells, although the specificity of these findings to a neutrophil population is undetermined (46). In other studies, coculture of neutrophils with T cells impaired Treg differentiation and promoted Th17 differentiation (17), leaving an open question as to whether neutrophils are predominantly immunosuppressive. In addition, it is unclear to what extent these in vitro assays recapitulate neutrophil–lymphocyte interactions in vivo. In particular, the results of in vitro culture of neutrophils are confounded by the fact that neutrophils are easily activated when isolated and cultured ex vivo, and thus neutrophil respiratory burst or neutrophil cell death in vitro may underlie the observed suppression of splenic lymphocyte proliferation. Our results significantly add to the literature by providing the first in vivo evidence, to our knowledge, for a dominant inhibitory effect for neutrophils in lupus with significant increases in Tfh and GC B cells upon early neutrophil depletion. These data are compatible with neutrophil inhibition of Tfh cell differentiation, but it is also possible that inhibition of B cell differentiation plays a role given the contacts between neutrophils and B cells throughout the disease course.

Our results highlight that the influence of neutrophils on adaptive immune dysregulation depends on both the specific cell encounter and the phenotype of the neutrophil. This may have parallels in other chronic inflammatory conditions such as HIV infection, cancer, and hepatitis B, where neutrophils can adopt a granulocytic myeloid-derived suppressor cell phenotype (13, 38, 39). In addition to ROS production, neutrophils may acquire a suppressive phenotype via production of a wide variety of soluble and cell surface mediators (4752). Our work expands the potential mechanisms by which neutrophils regulate adaptive immune responses in SLO, including TGF-β1, IL-1RA, and PD-L1. PD-L1 is particularly interesting in light of its association with a granulocytic myeloid-derived suppressor cell phenotype and a recent report demonstrating significant elevation in PD-L1–expressing neutrophils in human SLE, correlating with disease activity (53). PD-L1 delivers inhibitory signals to activated T and B cells upon ligation of PD-1, and thus may provide a critical negative feedback mechanism to dampen autoimmune responses. Of note, Tfh cells express PD-1, and PD-L1–PD-1 interactions have been specifically shown to negatively regulate Tfh cell expansion (54). Further, blocking PD-L1 in the NZB/W lupus mouse model increased splenic PD-1+ T cells, anti-DNA levels, and nephritis (55). Based on our data, we hypothesize that PD-L1 expression by splenic neutrophils and inhibition of Tfh cells may be a key mechanism by which neutrophils suppress lupus disease development.

In conclusion, our findings demonstrate the plasticity of neutrophil phenotype and function in terms of both transcriptional profile in SLO and changing spatial localization relative to B and T cells, dependent on the stage and severity of the disease. In turn, this neutrophil plasticity has dramatic effects on B cell autoimmunity, with surprisingly dominant regulatory effects as evidenced by the promotion of GC reactions and autoreactive PC generation in the setting of neutrophil depletion. Despite the apparent complexity of neutrophil contributions to the outcome of autoimmunity, it is increasingly clear that neutrophils can exert a strong influence on adaptive immune dysregulation in lupus, highlighting the need for further investigation into the factors that may tip the balance in neutrophil acquisition of a proinflammatory versus regulatory phenotype.

We thank the members of the University of Rochester’s Flow Cytometry Core and Genomics Research Center for technical expertise and helpful advice.

This work was supported by National Institutes of Health Grants P01-AI078907 and R01-AI-077674 (to J.H.A.), a Lupus Foundation of America LIFELINE Grant (to J.H.A.), and the Bertha and Louis Weinstein Research Fund (to J.H.A.). A.K.B. has been supported by a National Institutes of Health Clinical and Translational Science Award (5UL1TR000042-10) Trainee Pilot and a Center for Musculoskeletal Research Training Grant (2 T32AR053459). J.H.A. has also been supported by National Institute of Arthritis and Musculoskeletal and Skin Diseases Accelerated Medicines Partnership Grant 1UH2AR067690 (to J.H.A.).

The sequences presented in this article have been submitted to the Gene Expression Omnibus (https://www.ncbi.nlm.nih.gov/geo/) under accession number GSE97439.

The online version of this article contains supplemental material.

Abbreviations used in this article:

ASC

Ab-secreting cell

BAFF

B cell activation factor

FDR

false discovery rate

GC

germinal center

IHC

immunohistochemistry

NETosis

neutrophil extracellular trap formation

ROS

reactive oxygen species

SLE

systemic lupus erythematosus

SLO

secondary lymphoid organ

Tfh

T follicular helper

Treg

regulatory T cell.

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The authors have no financial conflicts of interest.

Supplementary data