Neutrophil-Derived IL-6 Potentially Drives Ferroptosis Resistance in B Cells in Lupus Kidney

Ferroptosis resistance is vital for B cell development, especially in inflammatory diseases, yet the underlying mechanism is still unclear. In this study, based on the scRNA-seq technique and flow cytometry, we discovered a proportion of neutrophils exhibited upregulated expression of the IL-6 and correlated with the expression of IL-6 receptor and SLC7A11 from B cells in lupus kidney. Moreover, we identified that in lupus kidney, neutrophils could provide IL-6 to facilitate ferroptosis resistance in B cells via SLC7A11, and inhibition of SLC7A11 could significantly enhance ferroptosis in B cells and could decrease B cell proliferation. This study helps understand the crosstalk between neutrophils and B cells in the kidney in the development of lupus.


Introduction
Ferroptosis is an iron-dependent form of regulated cell death characterized by the accumulation of lipid peroxides. Ferroptosis resistance, the ability of cells or tissues to evade ferroptotic cell death, has been implicated in the development of cell death and various diseases [1][2][3][4], yet its role in lupus still needs to be depicted. Lupus is an autoimmune disease characterized by hypergammaglobulinemia derived from pathogenic B cell behavior [5][6][7]. Illustrating the molecular mechanism of abnormal B cell, for example, the manner of B cell death, would help understand its role in driving the development of lupus.
It is known that B cell behavior is tightly regulated by the microenvironment [8,9], for example, neutrophils [10,11].
For example, in the spleen, neutrophil enters germinal center and provide BAFF, APRIL, IL-21, and PTX3 to stimulate immunoglobulin diversification and production [10,12]. In the lymph node, neutrophils appeared in B cell zone and potentially help activate, and prolong the life time of B cells by providing BAFF and APRIL [13]. However, whether neutrophil modulate B cell behavior and the potential molecular mechanism is still less discussed.
In this study, we demonstrated that a population of neutrophils helps B cells suppress ferroptosis via SLC7A11 in the lupus kidney. Strikingly, blocking IL-6 significantly promoted ferroptosis of B cells and showed its therapeutic effect. Precisely modulating IL-6-expressing neutrophils in the kidney would be a potential therapeutic strategy to improve clinical treatment for lupus.

Materials and Methods
2.1. scRNA-Seq Analysis. The scRNA-seq data of murine glomeruli including those suffering from glomerular nephritis (GN) was retrieved from the NCBI GEO database under the accession ID GSE146912 [14]. The R package Seurat (v4.3.0) was used to process scRNA-seq data, and cells were clustered at resolution 0.6 after removing those genes expressed by less than 3 cells and those cells with less than 200 genes sequenced; min:pct = 0:25 and logfc:threshold = 0:25 were used to identify enriched genes that were expressed by each cell types. The R package CellChat (v1.5.0) was used to analyze inferred cell-cell communications.
2.2. Mice. C57BL/6 mice were purchased from the Changsha Tianqin Ltd. All experimental mice were bred under SPF conditions and 12-hour day/12-hour night cycle. Only 10week-old male mice were used in this study. For induction of systemic lupus erythematosus, mice were introperitoneally injected with 500 μl of pristane (Sigma-Aldrich, #P2870) [15]. For the control group, mice were injected with an equal volume of PBS. All the procedures used in this study were approved by the Ethical Committee of Youjiang Medical University for Nationalities.

Flow Cytometry.
For preparation of a single cell suspension, freshly isolated kidney tissues were cut into small pieces, digested with Collagen IV (25 mg/mL, Gibco, #1704) at 37°C for 45 minutes, and filtered through a 100 μm stainless cell strainer. After blocking unspecific binding, samples were incubated with fluorescent-coupled antibodies. For intracellular staining, cells were fixed with 2% PFA for 15 minutes at room temperature, then permeabilized with permeabilization buffer (Invitrogen, #00-8333-56) at 4°C for 10 minutes, and incubated with fluorescent coupled antibodies. After washing twice with PBS/ 0.5%BSA, cells were resuspended in PBS and measured on the flow cytometer (Thermo Fisher Attune NxT). 2.6. Immunofluorescent Imaging. After removing the supernatant from cell culture, cells were fixed with 2% of paraformaldehyde (Sigma-Aldrich, #30525-89-4) at room temperature for 10 minutes, then cells were counterstained with DAPI (Invitrogen, #D1306), Lipid Peroxidation/Liper-Fluo kit or FerroOrange Kit according to the manufacturer's instructions. Immunofluorescent images were captured by the fluorescence microscope (Leica DMI300B). The fluorescent intensity was acquired by the software ImageJ.
2.8. Quantification of GSH and GSSG. Intracellular glutathione (GSH) and oxidized glutathione (GSSG) were quantitatively measured by a commercial kit (DOJINDO Laboratory, #G263). Briefly, B cells were harvested after culture and spun down at 4°C and 300 g for 10 min. After washing twice with cold PBS, 80 μl of 10 mM HCl solution was added to obtain lysate, then 20 μl of 5% SSA was added and spun down at 4°C, 8000 g for 10 min. The lysate was diluted in 0.5% SSA. GSH and GSSG were measured according to the manufacturer's instruction [16].
2.9. Statistical Analysis. All the data were statistically analyzed by Graphpad Prism 6.0. The nonparametric test was used to analyze the differences among groups. Data were presented as mean ± SD. p < 0:05 was considered statistically significant.

Neutrophil Heterogeneity in Lupus
Kidney. GN usually occurred in lupus. To explore the heterogeneity of neutrophils in glomeruli in GN kidney, we reanalyzed the scRNA-seq data [14]. After vigorous quality control (Supplementary Figure 1A, 1B, 1C and 1D), 6043 cells from healthy controls and 8181 cell from GN kidney were integrated for downstreaming analysis. In total, 27 cell clusters were identified based on enriched genes expressed by each cell type (Figure 1  To better explore the cell-cell communications between neutrophils and B cells in glomeruli suffering from GN, 3 neutrophil subpopulations and B cells identified from scRNA-seq data were isolated for downstreaming analysis (Figure 1(c)). Further comparison showed that the frequency of subcluster neutrophil (Ly6g) is much higher in lupus kidney ( Figure 1(d)), indicating this subpopulation plays an active role in modulating the microenvironment of GN. It is known that GN is also a pathological feature occurred to lupus. To validate and to check whether these 3 neutrophil subpopulations exist in lupus, freshly isolated lupus kidney biopsies were analyzed by flow cytometry, and these 3 neutrophil subclusters were identified (Figure 1(e)). Based on flow cytometry analysis, we found the subcluster neutrophil (Ly6g) in the lupus kidney increased by almost 30%, compared with healthy control kidney, the neutrophil (Aldh2) increased by 8%, and the number of neutrophil (Socs3) was similar to healthy ones ( Figure 1(f)). This suggested that neutrophil (Ly6g) could be the "main player" in the development of GN, comparing with the other two neutrophil subpopulations.

Neutrophil-Derived IL-6 Conducted a Major Signal between Neutrophils and B Cells in Lupus
Kidney. Isolated neutrophil subclusters and B cells were further analyzed with the R package CellChat [17] to quantitatively explore cellcell communications. As expected, the cell-cell interaction numbers between neutrophils and B cells were drastically increased, and the interaction strength was upregulated (Figures 2(a) and 2(b)). The increased signals were mainly distributed between neutrophil (Ly6g) and B cells ( Figure 2(b)). In lupus kidney, the signals derived from neutrophil (Ly6g) were significantly increased, while those derived from neutrophil (Socs3) were decreased, and the signals derived from neutrophil (Aldh2) seem similar to healthy controls (Figure 2(c)). Then, the signals between neutrophil (Ly6g) and B cells were examined and compared. Among which, IL-6 derived from neutrophil (Ly6g) "rise" to be the major signal interacting with B cells in the lupus kidney ( Figure 2(d)). Consistently, the genetic expression pattern showed IL-6 was mainly produced by neutrophil (Ly6g), and B cell significantly express the receptor for IL-6 (Supplementary figure 3) in GN. Furthermore, flow cytometry analysis of the lupus kidney showed the main source of IL-6 was expressed by neutrophil (Ly6g) (Figure 2(e)), and the statistical analysis showed that  Mediators of Inflammation neutrophil (Ly6g) should be the main "contributor" for providing IL-6, rather than the other 2 subpopulations, in the lupus kidney (Figures 2(e) and 2(f)).

Neutrophil-Derived IL-6 Induces Ferroptosis Resistance in Lupus Kidney B Cells.
To explore the inferred pathways in B cells that are inducted by neutrophil-derived IL-6, we performed a correlation test between IL-6 and the altered gene expression level in B cells. Strikingly, the B cellderived IL-6R, STAT3, and SLC7A11 positively correlated to neutrophil-derived IL-6 ( Figure 3(a)). It has been proven that the IL-6/STAT3/SLC7A11 pathway could conduct ferroptosis resistance [16,18,19]. In order to examine the potential role of neutrophilderived IL-6 in regulation of suppressing ferroptosis in lupus B cells, we performed coculture of B cells and neutrophil isolated from both healthy and lupus kidney. As expected, blocking IL-6 significantly increased the Fe 2+ signals (Figures 3(b) and 3(c)) and lipid peroxidation (Figures 3(d) and 3(e)) in B cells isolated from lupus animal, together with altered GSH and GSSG/GSH ratio (Figures 3(f) and 3(g)), key indicator for ferroptosis [20,21] but had no obvious effect on B cells isolated from healthy controls, suggesting IL-6 could suppress ferroptosis/ferroptosis resistance in lupus B cells.
To confirm this hypothesis, ferrostatin 1, an inhibitor of erastin-induced ferroptosis [22], was added into coculture experiments, and the above phenomena were significantly rescued (Figures 3(b) figure 4F) in B cells, indicating IL-6 should be the "main player" derived from neutrophils mediating ferroptosis resistance.
Considering B cells in the lupus kidney exhibited higher expression of SLC7A11 (Supplementary figures 5A and 5B), a downstreaming molecule mediated by the IL-6 in mediating ferroptosis resistance [16]. We treated the cocultured B cells and neutrophils with sulfasalazine (an inhibitor of SLC7A11 [21,23]). After sulfasalazine, the expression level of SLC7A11 was significantly downregulated (Supplementary figure 6), the Fe 2+ signal (Figures 3(h) and 3(i)) and lipid peroxidation (Figures 3(j) and 3(k)) was significantly increased in B cells isolated from lupus mouse, meanwhile the intracellular GSSH and GSH were also  10 Mediators of Inflammation altered accordingly (Figures 3(l) and 3(m)), indicating IL-6 facilitates ferroptosis resistance in lupus B cells via SLC7A11. Furthermore, the proliferation/mitosis of B cell were examined. Flow cytometry analysis showed B cell proliferation/mitosis was significantly decreased by blocking SLC7A11 (Figures 3(n) and 3(o)).

Inhibition of SLC7A11 Enhanced Ferroptosis of Kidney B
Cells in Lupus Mice. In order to examine whether IL-6 could conduct ferroptosis resistance in lupus kidney B cells via SLC7A11 in vivo, we treated lupus animals with sulfasalazine and found the Fe 2+ -enriched B cell numbers were drastically increased after blocking SLC7A11 (Figures 4(a)-4(c)), indicating SLC7A11 could be the key factor that drives B cells to develop ferroptosis, induced by IL-6, the cytokine mainly expressed by neutrophil (Ly6g). Moreover, B cell proliferation/mitosis was also measured by counterstaining Ki67, an antibody for measuring cell proliferation/mitosis [23]. Flow cytometry analysis exhibited that after blocking SLC7A11, the B cells proliferation/mitosis was significantly decreased (Figures 4(d) and 4(e)), indicating that blocking SLC7A11 could be a potential method to suppress B cells in lupus kidney.

Discussion
Both ferroptosis and ferroptosis resistance have been observed in studies on renal diseases. Muller et al. confirmed the existence of ferroptosis in acute kidney injury [24]; the study from Schreiber et al. was consistent with that ferroptosis could contribute to the development of polycystic kidney diseases [25,26]; and Miess et al. reported ferroptosis resistance in clear cell renal cell carcinoma [26,27]. However, the ferroptosis resistance in lupus is still less discussed. This study helps narrow the gap in understanding the role of ferroptosis/ferroptosis resistance in lupus.
Ferroptosis resistance has been shown to be involved in the pathogenesis of various diseases. A study by Huang et al. investigated the role of UBIAD1, an enzyme that was previously identified as an apoptosis mediator; however, it could significantly alleviate ferroptosis cerebral reperfusion insult [28]. Another study by Wang et al. showed that Wnt/beta-catenin signaling, usually considered proinflammatory, could facilitate ferroptosis resistance by targeting GPX4 in gastric cancer [29]. These studies proposed that both apoptotic factors and proinflammatory factors could exert ferroptosis resistance.
IL-6, known as a proinflammatory factor, has been proven to participate in the regulation of ferroptosis [30]. It has been reported that IL-6 could promote ferroptosis in goat mammary epithelial cells via NRF2 signaling [31] and promote bronchial epithelial cells by inducing ROS-   [16]. This study identified that IL-6 could protect B cells from ferroptosis in lupus kidney via the IL-6/STAT3/SLC7A11 axis. Taken together, these studies highlighted the complex role of IL-6 in regulating ferroptosis and suggested that the effect of IL-6 on ferroptosis may be context-dependent. Previous studies have reported neutrophils could modulate B cells [33,34] by providing BAFF, APRIL, which help activate B cells and even drive the antibody class switch and generation of IgG2 and IgA [34]. This study, however, discovered that a population of neutrophil could provide IL-6 for B cells and suppress ferroptosis in lupus kidney, via SLC7A11. This discovery would improve the understanding of the cross-talk between neutrophils and B cells and help provide an alternative strategy for suppressing the pathological features of B cells in lupus [35,36].
The scRNA-seq data analyzed in this study was obtained from surgically isolated murine glomeruli from murine in the development of GN induced by injury, not from the whole kidney, and GN is different from lupus; however, the downstreaming cell-cell communication analysis between neutrophils and B cells and the coculture experiment showed consistent result that neutrophil-derived IL-6 could promote ferroptosis resistance in lupus kidney B cells, suggesting this could possibly be a general phenomenon in the development of glomerular injury. Besides, the morphological feature of mitochondria should be further tested to solidify the status of ferroptosis, and genetic silencing/ knockout of SLC7A11 in B cells should be performed and provided to rule out off-target effects independent of SLC7A11.
This study indicated that IL-6 derived from neutrophils could locally modulate the ferroptosis resistance in B cells in the lupus kidney, mainly via SLC7A11. This research shed lights in delineating the microenvironment around B cells and provided a candidate solution for improving therapeutics against lupus.

Data Availability
The datasets and code generated or analysed in this study are available from the corresponding author upon reasonable request. with neutrophils. Each dot represents one readout. Data represents similar results from at least 3 independent experiments. * * p < 0:01; NS: no significance. Supplementary