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. 2025 Aug 4;48(6):4564–4578. doi: 10.1007/s10753-025-02347-9

Exposure to Dietary Nitrite Exacerbates Lupus in MRL/lpr Mice by Enhancing Integrin Alpha M

Yiwu Qiu 1, Qingyi Zhang 1, Xueting Yang 1, Chengping Wen 1, Zhixing He 1,✉, Mingzhu Wang 1,✉
PMCID: PMC12722331  PMID: 40758225

Abstract

There is a recognized longitudinal association between serum nitrogen oxides levels and disease activity in lupus nephritis. Recently, increased exposure to high levels of nitrite has raised significant concerns, though its impact on lupus pathogenesis has not been fully elucidated. Using the MRL/lpr spontaneous lupus model, we employed integrated immunological and transcriptomic approaches to investigate nitrite’s effects. Flow cytometry revealed significant elevations in splenic double negative T (DN T) cells, T follicular helper (Tfh) cells, and plasma cells following nitrite intervention, along with a reduction in splenic regulatory T (Treg) cells. ELISA quantification revealed elevated serum anti-double-stranded DNA antibodies (anti-dsDNA), antinuclear antibodies (ANA), and pro-inflammatory cytokines (IL-12p70, TNF-α), correlating with aggravated renal pathology in nitrite-exposed mice. Transcriptome analysis further revealed significant gene expression changes in both spleen and kidney tissues associated with nitrite exposure. Notably, three KEGG pathways, cell adhesion molecules, osteoclast differentiation, and B cell receptor signaling pathway, were consistently enriched in both the spleen and kidney transcriptomes. Subsequent western blot analysis identified that the Itgam (integrin alpha M)-related cell adhesion molecule pathway potentially mediated the mechanism by which nitrite aggravated lupus in MRL/lpr mice. To explore this mechanism, the integrin antagonist lifitegrast was used to inhibit the expression of Itgam in the nitrite-exposed MRL/lpr mice, resulting in attenuation of nitrite-induced lupus exacerbation. Collectively, these findings suggested that nitrite exposure could aggravate lupus by promoting Itgam expression.

Graphical Abstract

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Supplementary Information

The online version contains supplementary material available at 10.1007/s10753-025-02347-9.

Keywords: Nitrite, Systemic lupus erythematosus, Transcriptome, Cell adhesion molecules, Integrin alpha M

Introduction

Nitrite (NO2−), an inorganic compound with multifaceted applications, serves dual roles as a critical food preservative and a pervasive environmental contaminant [1]. Its widespread use in cured meat processing and agricultural practices has raised concerns about human exposure risks. The World Health Organization recommends a daily safety limit of 0.07 mg/kg, yet dietary intake accounts for ~ 80% of total exposure, primarily through processed foods [2, 3]. Concurrently, excessive fertilizer use and improper waste disposal have amplified environmental nitrite contamination, particularly in groundwater systems near intensive farmlands [4, 5]. Previous studies have demonstrated that the health effects of nitrites are dose-dependent [6–8]. Chronic consumption of low dose or acute exposure to high dose of nitrite can lead to organ dysfunction and the development of serious diseases [9, 10]. Emerging evidence further links nitrite toxicity to systemic inflammatory pathways, including gut dysbiosis and immune dysregulation [11–14]. Therefore, given its potential impact on systemic health, nitrite exposure warrants considerable attention.

Systemic lupus erythematosus (SLE) is a prototypical autoimmune disease that affects multiple organs in the human body [15]. The pathogenesis of SLE involves the triggering of T cell dysfunction by autoantigens, leading to abnormal B cell responses and sustained autoantibody production [16]. Despite advances in understanding, the etiology of SLE remains largely unclear, with genetic mutations, hormonal imbalances, environmental exposures, and dietary habits identified as potential contributors to disease progression [17, 18]. Several chemical exposures have been suggested to trigger systemic inflammatory responses and are postulated as potential contributors to SLE development [19–21]. Recent studies have reported associations between nitrite exposure, either from food or water, and immune-related diseases [22–24]. Additionally, longitudinal studies have reported positive associations between serum nitrite/nitrate levels and markers indicative of SLE and lupus nephritis disease activity [25]. Nevertheless, the specific relationship between nitrite exposure and SLE has not yet been explored.

This study employs the MRL/lpr murine model—a validated surrogate for human SLE—to investigate nitrite’s role in lupus progression [26]. Our findings demonstrate that nitrite exacerbates proteinuria and glomerular IgG deposition, mirroring lupus nephritis pathology in humans. Transcriptomic profiling of spleen/kidney tissues and functional validation of Itgam were performed to unravel underlying mechanisms. These results provide preclinical evidence for revising dietary guidelines in SLE management and emphasize the imperative to mitigate nitrite exposure in susceptible populations.

Methods

Animals and Chemicals

Eight-week-old MRL/lpr mice were procured from Shanghai SLAC Laboratory Animal Co., Ltd. Female MRL/lpr mice were selected for this study due to the higher prevalence of SLE in females. This murine model demonstrates clinical characteristics that more closely recapitulate human disease manifestations and exhibits more stable disease progression [27]. All animal experiments were conducted in compliance with the guidelines set forth by the Institutional Animal Care and Use Committee of China. After a one-week acclimatization period in our animal facility, the mice were randomly assigned to different experimental groups. The animals were maintained under controlled conditions, with a constant temperature of 25 ± 1 oC, a 12-hour light/dark cycle, and ad libitum access to food. The mice were provided with a standard laboratory diet throughout the duration of the study.

Sodium nitrite (purity ≥ 99.0%, Sigma-Aldrich, 7632-00-0) was administered via drinking water from 9 to 20 weeks of age. Body weights of the mice were recorded weekly. Additionally, lifitegrast (MedChemExpress, HY-19344 A) was administered via intraperitoneal injection once weekly, from 16 to 20 weeks of age.

Experimental Scheme and Sample Collection

The animal experiment in this study consisted of two parts. The first part aimed to investigate the effects of nitrite on MRL/lpr mice. Based on the findings from the first part, the second part of the experiment focused on Itgam to further explore the impact mechanisms of nitrite on lupus.

Experiment I

This experiment aimed to assess the impact of sodium nitrite on lupus development in MRL/lpr mice. The mice were randomly divided into three groups (n = 7 per group): [1] model (MT) group: received free access to drinking water; [2] low-dose nitrite (Low) group: received free access to a 0.1 g/L sodium nitrite solution, with the solution replaced every two days; [3] high-dose of sodium nitrite (High) group: received free access to a 0.3 g/L sodium nitrite solution, with the solution replaced every two days. The experiment was conducted over a 12-week period. Urine samples were collected every two weeks from 12 to 20 weeks of age. Blood samples were collected from the retro-orbital vein and centrifuged at 3000 rpm for 15 min to obtain serum. At 20 weeks of age, the mice were euthanized, and spleen and kidney tissues were harvested. The spleens were immediately weighed and processed into cell suspensions for flow cytometry analysis. One portion of the kidney tissue was snap-frozen in liquid nitrogen and stored at −80 oC, while the other portion was fixed in formaldehyde.

Experimental II

The second part of the study aimed to explore the impact pathogenesis of nitrite on lupus, with a particular focus on integrin alpha M. The MRL/lpr mice were divided into three groups (n = 7 per group): [1] model group: received free access to drinking water; [2] nitrite group: received free access to a 0.3 g/L sodium nitrite solution, with the solution replaced every two days; [3] lifitegrast group: received free access to a 0.3 g/L sodium nitrite solution, and from 16 to 20 weeks of age, mice were administered an intraperitoneal injection of lifitegrast at a dose of 15 mg/kg once weekly. The experiment lasted 12 weeks. Urine samples were collected every two weeks from 14 to 20 weeks of age. Blood samples were obtained from the retro-orbital vein and centrifuged at 3000 rpm for 15 min to obtain serum. At 20 weeks of age, the mice were euthanized to harvest tissues. The spleen was weighed and processed into cell suspensions for flow cytometry analysis. One kidney tissue sample was snap-frozen in liquid nitrogen and stored at −80 oC, while the other was fixed in formaldehyde for further analysis.

Urinary Protein Measurements

To monitor dynamic changes in urinary proteins, urine samples were collected biweekly from each experimental mouse starting at 12 weeks of age. Sample collection was performed using a standard metabolic cage system to ensure uncontaminated, spontaneous urine acquisition. Urinary protein concentrations were quantified using the Bradford Protein Assay Kit (Beyotime, P0006), following the manufacturer’s recommended protocols to maintain measurement consistency and reliability.

Flow Cytometry Analysis

Mouse spleens were homogenized and passed through a 70-mesh filter. Erythrocytes were lysed using an erythrocyte lysis solution, and the lysis was subsequently terminated. Single-cell suspensions were then prepared by adding RPMI 1640 culture medium, and the cell concentration was adjusted to 1 × 10^6 cells/mL. The experiment included blank control tubes, single-staining tubes, and sample tubes. Cells were washed with 1 mL staining buffer, centrifuged, and the supernatant was discarded. A total of 0.5 µL of Fc block antibody was added to all tubes and incubated for 15 min to prevent nonspecific binding. Following incubation, cells were washed again with 1 mL staining buffer, centrifuged, and the supernatant was discarded. For staining, antibodies were added as follows: CD3 (Biolgend, 100204), CD4 (Biolgend, 100412), CD8 (Biolgend, 162308), Bcl-6 (Biolgend, 358512), and CXCR5 (Biolgend, 145511) for the T cell staining panel, and CD19 (Biolgend, 152404), B220 (Biolgend, 103212), and CD138 (Biolgend, 142504) for the B cell staining panel. CD4 (Biolgend, 100204), CD25 (Biolgend, 113704), FOXP3 (Biolgend, 320014) for the Treg cell staining panel. Each of these antibodies was added to the appropriate sample tube, which was then incubated for 20 min. After incubation, cells were washed with staining buffer, centrifuged at 350 g for 5 min, and the supernatant was removed. Finally, cells were resuspended in 250 µL of cell staining buffer and analyzed using the Beckman CytoFLEX S flow cytometer.

Enzyme-Linked Immunosorbent Assay (ELISA)

The levels of anti-dsDNA antibodies and ANA were measured using enzyme-linked immunosorbent assay. The assays were performed according to the manufacturer’s instructions using CUSABIO ELISA Kits, which employ a double antigen sandwich ELISA method. Additionally, TNF-α and IL-12p70 levels were determined using Multisciences ELISA Kits.

Renal Histology and Immunofluorescence

Mouse kidney tissues were fixed in a formaldehyde solution for 2 days, followed by dehydration, paraffin embedding, and sectioning. The sections were treated with HD constant staining pretreatment solution, stained with hematoxylin, and then rinsed under running water. Subsequently, sections were immersed in eosin dye. After dehydration and mounting, images were acquired and analyzed.

For immunofluorescence staining, the procedure was carried out at room temperature. Paraffin-embedded kidney sections were deparaffinized and rehydrated, followed by antigen retrieval. Hydrogen peroxide was used to block endogenous peroxidase activity, and a hydrophobic barrier was drawn around the tissue using a histochemical pen. The tissue was then blocked with serum and incubated with the primary C3 antibody (1:500, Thermo PA1-29715). Subsequently, the corresponding HRP-labeled secondary antibody (1:500, Servicebio GB23303) was applied, followed by the addition of the appropriate TSA dye. The tissue sections were placed in an antigen retrieval buffer for additional antigen retrieval steps. The IgG antibody (1:2000, Proteintech, 30000-0-AP) was then introduced, followed by the corresponding secondary antibody (1:500, Servicebio GB21303). The cell nuclei were counterstained with DAPI to minimize autofluorescence, and the sections were sealed. Finally, images were captured for analysis.

Transcriptome Analysis

Total RNA was extracted from spleen or kidney tissues using TRIzol® Reagent, following the manufacturer’s instructions. RNA quality was assessed using the 5300 Bioanalyzer (Agilent), and quantification was carried out with the ND-2000 spectrophotometer (NanoDrop Technologies). RNA purification, reverse transcription, library construction, and sequencing were conducted by Shanghai Majorbio Bio-pharm Biotechnology Co., Ltd. (Shanghai, China), according to the manufacturer’s protocols (Illumina, San Diego, CA) as previously described [28]. The raw sequencing reads have been deposited in the NCBI Sequence Read Archive (SRA) under Accession Number PRJNA1165911.

The raw paired-end reads were processed using fastp [29] for trimming and quality control, employing default parameters. Clean reads were then aligned to the reference genome using HISAT2 [30] with orientation mode. The mapped reads for each sample were assembled using StringTie [31] based on a reference-guided approach. Differential expression genes (DEGs) between different samples were identified by calculating the expression level of each transcript using the transcripts per million reads (TPM) method. Gene abundance quantification was performed with RSEM [32]. Differential expression analysis was conducted using DESeq2 [33], with criteria set to|log2FC| ≥ 2 and a false discovery rate (FDR) ≤ 0.05. Furthermore, KEGG pathway enrichment analysis was carried out to identify DEGs significantly enriched in metabolic pathways, using KOBAS [34] with a Bonferroni-corrected P-value ≤ 0.05, compared against the whole-transcriptome background. To clarify the expression of interferon (IFN) genes, we analyzed changes in IFN gene expression in the spleen and kidney after exposure to nitrite using transcriptomic data.

Western Blot Analysis

The expression of Itgam and Cadm1 was also assessed via Western blot analysis. Kindey samples (50 mg) were homogenized in 9 volumes (v/w) of RIPA buffer (Beyotime, P0013B) supplemented with protease inhibitor (Beyotime, P1005) to extract total protein. The total protein concentration was determined using the BCA protein assay kit (Beyotime, P0010S). Forty micrograms of protein from each sample were loaded onto a 10% SDS-PAGE and transferred to a polyvinylidene difluoride (PVDF) membrane. The membrane was blocked with 5% skim milk powder in Tris-buffered saline containing 0.1% Tween-20 for one hour at room temperature, followed by overnight incubation with diluted primary antibodies against GAPDH (1:2000, Cell Signaling Technology, 5174 S), Itgam (1:1000, ABclonal A24120), or Cadm1 (1:1000, Thermo PA5-24196) at 4 °C. After incubation with the appropriate secondary antibody (1:10000, LI-COR, C50331-05) for 2 h at room temperature, the bands were scanned using an infrared imaging system (Odyssey, LI-COR) and quantified using ImageJ software.

Statistical Analysis

Statistical analyses were conducted using GraphPad Prism 8.0. Each experiment included a minimum of three biological replicates. Data were presented as mean ± standard deviation (± s). Parametric statistical methods were used for normally distributed data, while non-parametric tests were applied when the data did not meet this criterion. For comparisons across multiple groups, one-way analysis of variance (ANOVA) was employed. A p-value of < 0.05 was considered to indicate statistical significance.

Results

Effects of Nitrite on Lupus Symptoms

Similar to SLE patients, MRL/lpr mice exhibits lupus symptoms associated with systemic inflammation and renal injury. As shown in Fig. 1a and d, both low and high doses of nitrite exposure resulted in a significant increase in serum autoantibodies (anti-dsDNA and ANA) and pro-inflammatory cytokines (TNF-α and IL-12p70) in MRL/lpr mice. Additionally, nitrite exposure led to a marked elevation in urinary protein levels at 18 and 20 weeks old (Fig. 1e). Histopathological analysis using H&E staining revealed that nitrite exposure exacerbated kidney dysfunction, as evidenced by increased pathological score, glomerular crescent formation, endocapillary proliferation, and inflammatory infiltration (Fig. 1f and g). Moreover, kidney injury was strongly correlated with immune complex deposition and impaired complement system function. As illustrated in Fig. 1h and j, there was a significant increase in IgG deposition (green) and C3 expression (red) in the nitrite-treated groups compared to the model group. Overall, these findings suggest that nitrite exposure could exacerbate lupus symptoms in MRL/lpr mice.

Fig. 1.

Fig. 1

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Effects of nitrite exposure on lupus symptoms in MRL/lpr mice. (a-d) Serum levels of anti-dsDNA, ANA, TNF-a, and IL-12p70 in MRL/lpr mice were assessed in MRL/lpr mice. (e) The urinary protein levels were measured in MRL/lpr mice from 12 to 20 weeks of age. (f, g) Representative images of kidney sections stained with H&E are presented, alongside pathological scores for kidney tissue. Black arrows indicate areas of glomerulonephritis and inflammatory infiltrates. (h) Immunofluorescence staining was performed to visualize C3 deposition (red) and IgG accumulation (green) in kidney sections, and the objective magnification was 40×. The white arrow indicates the glomerulus. (i, j) The mean fluorescence intensity of C3 and IgG accumulation was quantified using ImageJ software. Asterisks denote statistical significance: “*” for p < 0.05, “**” for p < 0.01, and “ns” indicates no significance. MT, MRL/lpr mice treated with normal water (n = 6); Low, MRL/lpr mice treated with low-dose nitrite solution (n = 7); High, MRL/lpr mice treated with high-dose nitrite solution (n = 7).

Effects of Nitrite on the Percentage of Immune Cells in Spleen

Splenomegaly is recognized as a characteristic feature of active lupus. In this study, nitrite exposure at both doses resulted in a significant increase in the spleen index (spleen weight/body weight) in MRL/lpr mice (Fig. 2a and b, Supplementary Fig. 1). Given that T and B cells in the spleen are crucial mediators in the pathogenesis of SLE, notable changes were observed following nitrite exposure. Specifically, nitrite exposure led to a marked increase in the percentage of DN T cells, Tfh cells, and plasma cells in the spleens of MRL/lpr mice (Fig. 2c, f, i and j, Supplementary Fig. 2). Flow cytometry gating strategies are demonstrated in Supplementary Figs. 3, 4. Conversely, the percentage of Treg was significantly reduced in response to nitrite exposure, suggesting an imbalance in immune regulation associated with lupus pathogenesis (Fig. 2g and h).

Fig. 2.

Fig. 2

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Effects of nitrite exposure on spleen immune cells in MRL/lpr mice. (a, b) Representative images of spleens and the spleen index under different treatment conditions are presented. (c-j) Flow cytometry analysis images and the mean abundance of DN T, Tfh, Treg, and plasma cells in spleen are displayed. Asterisks indicate statistical significance: “*” for p < 0.05, “**” for p < 0.01, and “ns” indicates no significance. MT, MRL/lpr mice treated with normal water (n = 6); Low, MRL/lpr mice treated with low-dose nitrite solution (n = 7); High, MRL/lpr mice treated with high-dose nitrite solution (n = 7).

Effects of Nitrite on Spleen and Renal Transcriptome

Both low and high doses of nitrite exposure induced phenotypic damage in the spleen and kidneys of MRL/lpr mice. To further elucidate the mechanisms underlying nitrite-induced exacerbation of lupus, transcriptome analysis was conducted on spleen and kidney tissues from the model group and the high-dose nitrite group. A total of 505 DEGs were identified in spleen tissue, with 335 upregulated and 170 downregulated in the high-dose nitrite group compared to the MT group (Fig. 3a). These 505 DEGs were significantly enriched in three KEGG pathways: cell adhesion molecules, osteoclast differentiation, and B cell receptor signaling pathway (Fig. 3b).

Fig. 3.

Fig. 3

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Effects of nitrite exposure on spleen and kidney transcriptome. (a) The volcano plot illustrates the p-values and log fold changes of spleen genes between the MT and high nitrite groups, red dots represent upregulated genes in the high nitrite group, while light blue dots indicate downregulated genes in high nitrite group; (b) Functional annotation of differentially expressed spleen genes between the MT and high nitrite groups at KEGG level 3 is presented. (c) The volcano plot displays the p-values and log fold changes of kidney genes between the MT and high nitrite groups, with red dots representing upregulated genes and light blue dots indicating downregulated genes in the high nitrite group; (d) Functional annotation of differentially expressed kidney genes between the MT and high nitrite groups at KEGG level 3 is provided. MT, MRL/lpr mice treated with normal water; High, MRL/lpr mice treated with high-dose nitrite solution

In the kidney tissues of MRL/lpr mice, high-dose nitrite exposure resulted in 1245 DEGs (525 upregulated and 720 downregulated), which were enriched in eight KEGG pathways (Fig. 3c and d). Notably, three KEGG pathways, cell adhesion molecules, osteoclast differentiation, and B cell receptor signaling pathway, were commonly enriched in both spleen and kidney transcriptome analyses (Fig. 4a). In total, 17 DEGs from the spleen and 24 DEGs from the kidney were involved in the cell adhesion molecules pathway (Fig. 4b and c). Comparative analysis revealed that two genes, Itgam and Cadm1 (cell adhesion molecule 1), were common between the 17 DEGs set and the 24 DEG set (Fig. 4b and c).

Fig. 4.

Fig. 4

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Common variations in transcriptome between spleen and kidney tissues. (a) Common enriched KEGG pathways identified between spleen and kidney transcriptomes. (b, c) The heatmap illustrates the DEGs associated with the KEGG pathway “cell adhesion molecules” in both spleen and kidney tissues. (d) IFN-related gene expression. (e-g) Western blot analysis demonstrates the effects of nitrite exposure on the expression of Itgam and Cadm1 proteins expression in kidney tissues. “**” denotes p < 0.01, and “ns” indicates no significance. MT, MRL/lpr mice treated with normal water (n = 6); Low, MRL/lpr mice treated with low-dose nitrite solution (n = 7); High, MRL/lpr mice treated with high-dose nitrite solution (n = 7).

Type 1 IFNs play a central role in SLE pathogenesis, evidenced by dysregulated expression of IFN-stimulated genes in peripheral blood of affected patients. Approximately 50% of patients exhibit aberrant IFN pathway activation, with this gene signature correlating with severe organ involvement (e.g., renal, hematopoietic, and central nervous systems) [35, 36]. In this study, comparative analysis of splenic and renal tissues revealed significant upregulation of multiple IFN-related genes in both organs (Fig. 4d). Notably, Ifi7l2a was the sole gene demonstrating reduced expression, whereas all other interrogated IFN-associated genes showed elevated levels. These findings confirm that exposure to nitrite activates the IFN pathway in lupus target organs.

Further validation through western blot analysis demonstrated that high-dose nitrite exposure led to an upregulation of Itgam protein expression, whereas no significant effect was observed on Cadm1 protein (Fig. 4e and g). In summary, these findings suggest that the Itgam-related cell adhesion molecules pathway plays a pivotal role in the nitrite-induced exacerbation of lupus in MRL/lpr mice.

Lifitegrast Alleviate Effects of Nitrite on Lupus in MRL/lpr Mice

Lifitegrast, a novel small-molecule integrin antagonist, is known to block the interaction between intercellular adhesion molecule 1 and lymphocyte function-associated antigen 1 [37]. To elucidate the role of Itgam in lupus pathogenesis, this study utilized lifitegrast to treat MRL/lpr mice exposed to nitrite. In the first part of the experiment, although the effects of the two concentrations of nitrite were similar in terms of serum antibody and inflammatory factor levels, when renal injury was observed in hematoxylin and eosin sections, the glomerular injury was more severe in the field of view in the high-concentration nitrite group. There was a significant difference in the elevation of complement C3 levels; therefore, the high-concentration nitrite was chosen for the subsequent experiments.

As illustrated in Fig. 5a and f, lifitegrast treatment resulted in the inhibition of Itgam protein expression, accompanied by reductions in urinary protein and serum levels of anti-dsDNA, IL-12p70, and TNF-α in MRL/lpr mice. Additionally, lifitegrast administration led to a decrease in spleen index, as well as a reduction in DN T cells, Tfh cells, and plasma cells in the spleen, compared to nitrite-treated MRL/lpr mice (Figs. 5g and h and 6a and f, Supplementary Fig. 5). Lifitegrast reduced kidney injury and immune complex deposition. As shown in Fig. 5i and k, IgG deposition (green) and C3 expression (red) were significantly reduced in the lifitegrast group compared with the nitrite-treated group. Moreover, lifitegrast alleviated renal inflammatory cell infiltration and pathological scores (Fig. 6g and h). Collectively, these findings suggest that lifitegrast exerts an ameliorative effect, indicating that Itgam mediates the nitrite-induced exacerbation of lupus in MRL/lpr mice.

Fig. 5.

Fig. 5

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Effects of lifitegrast on lupus disease in MRL/lpr mice. (a, b) lifitegrast reduces the expression of Itgam protein in kidney tissue. (c-f) The effects of lifitegrast on urinary proteins levels and serum concentrations of anti-dsDNA, TNF-a, and IL-12p70. (g, h) The impact of lifitegrast on splenomegaly in MRL/lpr mice. (i) Immunofluorescence staining was performed to visualize C3 deposition (red) and IgG accumulation (green) in kidney sections, and the objective magnification was 40×. The white arrow indicates the glomerulus. (j, k) The mean fluorescence intensity of C3 and IgG accumulation was quantified using ImageJ software. Asterisks denote statistical significance: “*” denotes p < 0.05, “**” denotes p < 0.01, and “ns” indicates no significance. Model, MRL/lpr mice treated with normal water (n = 7); nitrite, MRL/lpr mice treated with high-dose nitrite solution (n = 7); lifitegrast, MRL/lpr mice treated with high-dose nitrite solution and lifitegrast (n = 7).

Fig. 6.

Fig. 6

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Effects of lifitegrast on spleen immune cells in MRL/lpr mice. (a-f) Flow cytometry analysis images depicting the mean abundance of DN T, Tfh, and plasma cells in the spleen. (g, h) Representative images of kidney sections stained with H&E, along with pathological scores of kidney tissue. “*” denotes p < 0.05, “**” denotes p < 0.01, and “ns” indicates no significance. Black arrows indicate areas of glomerulonephritis and inflammatory infiltrates. Model, MRL/lpr mice treated with normal water (n = 7); nitrite, MRL/lpr mice treated with high-dose nitrite solution (n = 7); lifitegrast, MRL/lpr mice treated with high-dose nitrite solution and lifitegrast (n = 7).

Discussion

Nitrite, while essential as a food preservative for its bacteriostatic and antioxidant properties [38], poses significant health risks at elevated exposure levels. Epidemiological studies associated dietary nitrite intake with SLE progression, including reduced complement 3 levels in frequent meat consumers [39] and correlations between serum nitrite/nitrate concentrations and lupus nephritis activity markers [40]. This study aims to elucidate the impact of nitrite on the progression of SLE using MRL/lpr murine model. Our study demonstrated that nitrite exposure dose-dependently to accelerate lupus progression in the MRL/lpr model, marked by disrupted splenic lymphocyte homeostasis (Treg reduction with DN T cell/Tfh cell expansion). This observation aligned with previous studies indicating that nitrite exposure can alter lymphocyte proliferation and differentiation in both humans [23] and mice [41].

Notably, exposure to nitrites resulted in significant splenomegaly. Although the number of DN T cells, Tfh cells, and plasma cells increased, accompanied by a decrease in Treg cells, this change was not sufficient to fully explain the threefold increase in splenic volume. Previous studies implicated that non-cellular components, such as venous congestion, erythrocyte retention [42, 43], hemolytic circulation triggered by erythrocyte destruction [44], and antibody overproduction [45], may contribute synergistically to splenomegaly. Additionally, nitrite exposure induced a significant increase in serum autoantibodies (anti-dsDNA and ANA) and inflammatory cytokines (TNF-α, IL-12p70) in MRL/lpr mice. Nitrite had demonstrated bidirectional effects on inflammatory cytokine production in prior literature [6, 46]. In terms of renal damage, nitrite exposure significantly exacerbated kidney injury, consistent with previous reports establishing a positive relationship between nitrite exposure and renal damage [47, 48].

Treg cells, as regulators of immune homeostasis, have been shown to inhibit the activation of effector T cells and macrophages by secreting IL-10 and TGF-β [49]. In this study, nitrite intervention reduced Treg cell numbers and exacerbated kidney injury in mice, indicating that Treg depletion leaded to uncontrolled effector immune cells and aggravated tissue damage. Nitrite-induced oxidative stress may generate reactive nitrogen intermediates that damage Treg DNA or mitochondria and promote apoptosis [50]. This is consistent with mutations in the Foxp3 gene leading to developmental defects in Treg cells and fatal autoimmune diseases, as well as with studies of Treg cells transplantation as a therapeutic strategy for autoimmune diseases [51–53].

Transcriptomic analysis identified three conserved pathways linking nitrite exposure to lupus exacerbation: cell adhesion molecules, osteoclast differentiation, and B cell receptor signaling. Cell adhesion molecules facilitate interactions between immune cells and endothelial cells, contributing to inflammation and tissue damage in SLE patients [54]. Osteoclast differentiation is closely associated with bone resorption and osteoporosis, common complications in SLE [55]. Meanwhile, dysregulated BCR signaling leads to the activation and survival of autoreactive B cells, which produce the autoantibodies characteristic of SLE [56]. These pathways may play a crucial role in the mechanism by which nitrite exacerbates lupus in MRL/lpr mice.

Among these pathways, “cell adhesion molecules”exhibited the most significant enrichment in both spleen and kidney tissues, suggesting their pivotal role in nitrite-induced lupus exacerbation. Furthermore, two genes—Itgam and Cadm1—were involved in this pathway and were upregulated by nitrite in both spleen and kidney tissues. Further studies showed that only Itgam changed at the protein level. Itgam, a subunit of the Mac-1 complex, is involved in immune cell adhesion and migration and contributes to the recruitment of leukocytes to inflamed tissues, thereby exacerbating chronic inflammation and tissue damage in SLE [57]. Variations at the Itgam gene, which encodes for the Mac-1 integrin, are one of the strongest genetic risk factors for SLE [58]. To further explore the role of Itgam in mediating the aggravating effect of nitrite on lupus, lifitegrast, a potent integrin antagonist, was used to treat MRL/lpr mice exposed to nitrite. Lifitegrast effectively alleviated nitrite-induced lupus symptoms in MRL/lpr mice, further suggesting that Itgam was involved in the pathogenesis of nitrite-aggravated lupus. Although lifitegrast’s clinical efficacy has been demonstrated in numerous previous studies [59, 60], this study highlighted its potential therapeutic role in lupus. This trial highlighted the potential therapeutic application of lifitegrast in alleviating lupus symptoms. However, challenges remain in clinical translation, including off-target effects and the clinical feasibility of targeting Itgam.

Beyond direct immune dysregulation, nitrite may indirectly exacerbate lupus via conversion to nitrosamines, known as carcinogens [61, 62]. Nitrosamines are crucial in preventing the body from effectively removing its own abnormal cells and antigens, which could exacerbate the development of autoimmune diseases [63, 64]. This hypothesis warrants further investigation.

This study has limitations: (1) The MRL/lpr model, while validated for SLE research, incompletely mirrors human disease complexity; (2) A key limitation is the absence of a lifitegrast-treated MRL/lpr control group to differentiate between drug-specific effects and nitrite-mediated impacts; (3) The mechanism of nitrite-induced Itgam upregulation remains uncharacterized. Further experiments complementing the effects of lifitegrast on MRL/lpr mice will lead to more robust conclusions. Future work should address these gaps through human cohort validation and mechanistic studies.

Conclusions

Our study established dietary nitrite as an environmental accelerator of lupus progression via Itgam. These findings advocated for dietary nitrite regulation in SLE management and highlight integrin antagonism as a potential therapeutic strategy.

Electronic Supplementary Material

Below is the link to the electronic supplementary material.

Supplementary Material 1 (DOCX 3.18 MB) (3.2MB, docx)
Supplementary Material 2 (PDF 226 KB) (227KB, pdf)

Acknowledgements

Not applicable.

Author Contributions

MW and ZH contributed toward conceiving the research. MW, QZ and XY conducted animal experiments. YQ assisted in conducting the experiments. HZ and YM analyzed the data, ZH and drafted the manuscript, CW, YM, and ZH revised the manuscript. All authors contributed to the article and approved the submitted version.

Funding

This work was supported by the National Natural Science Foundation of China (No. U21A20402, 82174208).

Data Availability

The RNA sequence data are accessible under accession no: PRJNA1165911. Further information and requests for resources and reagents should be directed to the corresponding author, M.W. (20231111@zcmu.edu.cn).

Declarations

Ethics Approval and Consent to Participate

All animal handling and experimental procedures were performed following local ethical committees and the National Institutes of Health Guide for the Care and Use of Laboratory Animals. All efforts were made to minimize animal suffering and to reduce the number of animals used. All procedures performed in this study involving animals were approved by the Ethics Committee of Zhejiang Chinese Medical University (Approval No. 20210621-08).

Consent for Publication

Not applicable.

Competing interests

The authors declare no competing interests.

Clinical Trial Number

Not applicable.

Footnotes

Yiwu Qiu, Qingyi Zhang, Xueting Yang are co-first authors; order is determined by relative overall contributions.

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Contributor Information

Zhixing He, Email: hzx2015@zcmu.edu.cn.

Mingzhu Wang, Email: 20231111@zcmu.edu.cn.

References

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary Material 1 (DOCX 3.18 MB) (3.2MB, docx)
Supplementary Material 2 (PDF 226 KB) (227KB, pdf)

Data Availability Statement

The RNA sequence data are accessible under accession no: PRJNA1165911. Further information and requests for resources and reagents should be directed to the corresponding author, M.W. (20231111@zcmu.edu.cn).

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