Comparison of TLR4, NF-κB and IRF3 expression in kidney tissue between lupus nephritis (LN) and systemic lupus erythematosus (SLE): a pristane-induced lupus mice model study

Abstract

Introduction and purpose

Lupus nephritis (LN) is a major cause of morbidity and mortality in patients with SLE, a complex autoimmune disease characterised by loss of tolerance to self-nuclear antigens. Toll-like receptor 4 (TLR4), the first line of defence in the innate immune system, has been linked to the pathogenesis of autoimmune diseases and LN by activating nuclear factor-κB (NF-κB) or interferon regulatory transcription factor 3 (IRF3). Local expression of those biomarkers in pristane-induced lupus mice is still unknown. Therefore, this study aimed to prove the role of TLR4, NF-κB and IRF3 in pristane-induced lupus mice.

Subjects and methods

The study subjects were female Balb/c pristane-induced lupus mice model, 8–12 weeks of age, n=30, divided into two groups, nephritis (LN group) and non-nephritis (SLE group). The control group were age-matched healthy female Balb/c mice, n=11. All mice were euthanised at weeks 16. Kidney tissue was taken for histopathology examination and TLR4, NF-κB, IRF3 immunofluorescence assay. The diagnosis of LN was based on proteinuria and histopathology examination according to the ISN/RPS 2004 classification of LN. Statistical analysis was performed using IBM SPSS Statistics V.25. P value <0.05 was considered statistically significant.

Results

There were significant differences in the expressions of TLR4, NF-κB and IRF3 among the LN, SLE and healthy control groups (p=0.000), with the highest expression found in the LN group for all markers. The linear regression between TLR4 and NF-κB resulted in p value=0.000; R²=0.817; β=0.904. Linear regression between TLR4 and IRF3 showed p value=0.000; R²=0.896; β=0.947, which means TLR4 had an 81.7% effect on NF-κB and 89.6% on IRF3 expression.

Conclusion

TLR4, NF-κB and IRF3 expression were increased in lupus, with the highest expression found in the LN group, suggesting that these biomarkers may be responsible for the development of nephritis in SLE, with TLR4 likely playing a dominant role in this pathway. Increased expression of these biomarkers in lupus without nephritis may indicate progression towards nephritis, which still needs to be proven with further research.

Correspondence to Loeki Enggar Fitri; [email protected]

WHAT IS ALREADY KNOWN ON THIS TOPIC

• Kidney injury involving toll-like receptor 4 (TLR4) can occur through the myeloid differentiation primary-response protein 88 (MYD88) pathway, which involves the activation of nuclear factor-κB (NF-κB), or through activation of the transcription factor interferon regulatory factor (IRF).

• Local expression of those biomarkers in the pristane-induced lupus mouse model is still unknown.

WHAT THIS STUDY ADDS

• TLR4, NF-κB and IRF3 expression were increased in lupus, with the highest expression found in the LN group, suggesting that these biomarkers might be responsible for the occurrence of nephritis in SLE, with TLR4 likely playing a dominant role in this pathway.

HOW THIS STUDY MIGHT AFFECT RESEARCH, PRACTICE OR POLICY

• This study is still a preliminary investigation that focuses on the role of TLR4/NF-κB and TLR4/IRF3 in LN.

• Future research could further explore the mechanistic pathways involving specific pro-inflammatory mediators that may link TLR4 activation to the progression of nephritis.

Introduction

SLE is a prototype of systemic autoimmune disease characterised by heterogeneous manifestations, multisystem involvement and production of various autoantibodies.¹ The prevalence and incidence of SLE have increased in recent years.² The kidney is the main target organ in SLE. Approximately 50% of patients with SLE have manifestations of lupus nephritis (LN), which is the main cause of morbidity and mortality.³ Patients with active LN have a poor long-term prognosis, with approximately 30% progressing to end-stage renal disease, requiring dialysis or kidney transplantation.^{1 4}

The immune mechanism underlying the occurrence of SLE/LN involves the presence of autoantibodies and impaired clearance of immune complexes (antigen-autoantibody).¹ Both the innate and adaptive immune systems are critically involved in misdirected immune responses to self-antigens.^{5 6} Evidence suggests that TLRs, which have previously been shown to be important for host defence, are involved in the pathogenesis of autoimmune diseases by recognising self-molecules.^7–9

Toll-like receptor 4 (TLR4) is a single transmembrane cell surface receptor within the pattern recognition receptor family, playing a key role in the innate immune system.^{7 10 11} Upregulation of TLR4 at the protein or gene level is a potent trigger for inducing lupus-like autoimmune diseases.⁷ Kidney injury involving TLR4 can occur through the myeloid-differentiation primary-response protein 88 (MYD88) pathway, which leads to the activation of nuclear factor-κB (NF-κB).¹¹ NF-κB is a key transcription factor in macrophages, regulating a large number of genes involved in various immune and inflammatory response processes, including the encoding of cytokines and chemokines.¹²

Kidney injury can also occur through a pathway independent of MYD88 (Toll/IL-1 receptor domain-containing adaptor protein/TIRAP), as TLR4 induction can activate the transcription factor interferon regulatory factor (IRF).¹² The IRF3 regulatory factor is a transcriptional regulator of cellular responses in various cell types and plays a crucial role in innate immunity.^{13 14} The local expression of these biomarkers in pristane-induced lupus mice is still unknown. Therefore, this study aimed to investigate the role of TLR4, NF-κB and IRF3 in pristane-induced lupus mice.

Materials and methods

Study design

This was an observational analytic study with cross-sectional design. The study was conducted at the Faculty of Medicine Universitas Brawijaya and Dr Saiful Anwar General Hospital Malang, July 2023 to September 2024.

Study subjects

The study subjects were female Balb/c (Mus musculus) lupus mice models, aged 8–12 weeks (n=30). The controls used were age-matched healthy female Balb/c (M. musculus) mice (n=11). The mice were obtained from Pusvetma, Surabaya, Indonesia. All animals were bred and housed at the Animal Research Laboratory (LPHC) of the Faculty of Medicine, Universitas Brawijaya, in controlled temperature rooms with light-dark cycles, and were provided with continuous access to food and water. The lupus mice groups were induced with a single intraperitoneal injection of 0.5 cc of pristane. In the control group, 0.5 cc of phosphate-buffered saline (PBS) was injected intraperitoneally. All mice were euthanised by CO₂ inhalation at week 16 after the administration of pristane and PBS injections. Kidney tissue was collected for kidney pathology examination and immunofluorescence assays of TLR4, NF-κB and IRF3.

Histopathological assessment for LN

The kidneys were fixed in 10% formalin solution and embedded in paraffin using the a Tissue-Tek Processor (Tissue-Tek, Torrance, California, USA). The specimens were then cut into 3–5 µm slices using a microtome (Leica 2245 RM, Microsystems (SEA), Ayer Rajah Industrial Estate, Singapore) and stained with H&E, Periodic Acid-Schiff, Masson’s trichrome and Jonas Methenamine Silver (Atom Scientific, Hyde, Cheshire, England). The histopathological slides were evaluated under light microscopy by two pathologists. The biopsies were classified according to the ISN/RPS 2004 classification of LN. Class I is characterised by normal glomeruli; class II shows pure mesangial hypercellularity to varying degrees or mesangial matrix expansion with mesangial immune deposits. Both class III and class IV reveal active or inactive focal endocapillary or extracapillary glomerulonephritis, either segmental or global. However, class III indicates that <50% of the glomerulus is involved, whereas class IV indicates that 50% or more of the glomerulus is involved. Class V is characterised by subepithelial immune deposits, either global or segmental, or their morphological sequelae, with or without mesangial changes. Based on the results of the histopathology examination, the mice were then grouped into healthy control, SLE group (non-nephritis), LN group (LN class I–V).

Immunofluorescence for TLR4, NF-κB, IRF3

The tissue slides were heated at 60°C for 60 min, then soaked in xylol (2×10 min), absolute ethanol (2×10 min), 90% ethanol (1×5 min), 80% ethanol (1×5 min), 70% ethanol (1×5 min) and sterile aquadest (3×5 min). The slides were washed with PBS for 3×5 min, followed by PBS containing 0.2% Triton-X 100 for 5×1 min. Then, the slides were incubated with 3% bovine serum albumin (BSA) for 30 min at room temperature. The BSA solution was discarded, and the slides were incubated with primary antibodies: IRF3 (FITC IRF-3(D-3):sc-376455, Santa Cruz Biotechnology, Santa Cruz, California, USA) at a 1:100 dilution, TLR4 (PE TLR4(MTS510):sc-13591, Santa Cruz Biotechnology) at a 1:100 dilution and NF-ĸB p50 (AlexaFluor405 NF-ĸB p50(E-10):sc-8414, Santa Cruz Biotechnology) at a 1:100 dilution. The slides were incubated overnight at 4°C. The slides were washed with PBS for 3×5 min, then covered with mounting medium and a cover glass. Observation was carried out using a fluorescence microscope. Expression analysis of each marker was performed using ImageJ 1.53c.

Statistical analysis

Statistical analysis was performed using IBM SPSS Statistics V.25. The analysis of expression differences among the healthy control, SLE and LN groups was carried out using one-way analysis of variance (ANOVA). Further analysis of expression differences between LN and healthy controls, SLE and healthy controls and SLE and LN groups was conducted using post hoc tests. Pearson’s correlation tests were used to assess the relationship between TLR4 and NF-κB, as well as between TLR4 and IRF3. Linear regression was used to determine how one variable impacts another or how changes in one variable induce changes in another. A p value of <0.05 was considered statistically significant.

Results

Ten mice (30.00%) from 30 pristane-induced lupus mice developed SLE, while 20 mice developed LN. The histopathological findings are shown in figure 1 and table 1. Expression analysis of TLR4, NF-κB and IRF3 was performed using ImageJ 1.53c and presented as percentage (%) (figure 2).

Table 1

TLR4, NF-κB and IRF3 expression among healthy controls, SLE and LN groups

	Healthy control N=11	SLE N=10	LN N=20	P value
LN class
I			0 (0.00%)
II			2 (10.00%)
III			6 (30.00%)
IV			2 (10.00%)
III and V			3 (15.00%)
IV and V			7 (35.00%)
TLR4 expression (%)				0.000
Mean+SD	27.63±3.07	37.55±3.10	51.20±5.04
Median	27.91	38.01	51.41
Range	22.82–31.05	33.50–42.68	43.92–61.71
NF-κB expression (%)				0.000
Mean+SD	23.18±7.22	32.60±3.28	45.94±5.23
Median	25.61	32.08	45.21
Range	10.46–31.94	28.82–40.03	39.16–54.72
IRF3 expression (%)				0.000
Mean+SD	23.79±5.28	34.06±2.50	46.68±5.86
Median	24.87	33.73	47.35
Range	15.49–32.28	28.42–37.71	34.79–55.45

SLE showed no nephritis; LN showed LN class I–V.

IRF3, interferon regulatory transcription factor 3; LN, lupus nephritis; NF-κB, nuclear factor kappa B; TLR4, toll-like receptor 4.

One-way ANOVA showed significant differences in TLR4 and IRF3 expressions among healthy controls, SLE and LN (p=0000). Kruskal-Wallis analysis showed significant differences in NF-κB expression among healthy controls, SLE and LN (p=0000) (table 1). Post hoc tests showed significant differences in TLR4 and IRF3 expressions between LN and healthy controls, SLE and healthy controls as well as SLE and LN, with a p value of 0.000 for all comparisons. Post hoc tests revealed significant differences in NF-κB expression between LN and healthy controls, SLE and LN (p=0000 and p=0001), but no difference between SLE and healthy controls (p=0086).

There was significant correlation between TLR4 and NF-κB (p=0.00, R=0.904), as well as correlation between TLR4 and IRF3 (p=0.00; R=0.947) (figure 3). Both had positive correlation with strong association.

Linear regression between TLR4 and NF-κB (p=0.000; R²=0.817; β=0.904) showed that the effect of TLR4 on NF-κB was 81.7%. Linear regression between TLR4 and IRF3 (p=0.000; R²=0.896; β=0.947) showed that the effect of TLR4 on IRF3 was 89.6%.

Discussion

TLR4 is a family of pattern receptors. This receptor can recognise molecular patterns associated with conserved pathogens, bind pathogen-associated molecular patterns from Gram-negative bacteria and serves as the first line of defence. TLR4 plays an important role as an amplifier of inflammation. A reduction in TLR4 expression significantly decreases the expansion of B lymphocyte cells and reduces ANAs.^{10 11 15} Lu et al found that C57BL/6 (lpr/lpr)-TLR4-deficient mice have a reduced number of marginal-zone B cells due to decreased expression of B lymphocyte stimulator receptors, which affects B cell maturation. TLR4 may also enhance autoantibody production through the increased expression of T helper (Th)1-related and Th17-related cytokines.¹⁵ The TLR4 pathway is likely involved in the pathogenesis of renal damage in LN through its binding to DNA-binding protein, high mobility group box 1 protein (HMGB1) and lupus autoantigens.^{7 16}

In the kidney, TLR4 is expressed by parenchymal cells and by infiltrating neutrophils and mononuclear phagocytes, including macrophages and dendritic cells. Mesangial cells and podocytes also express TLR4 in the nephritic environment. Mesangial cells isolated from NZB/W mice with spontaneous autoimmunity express significantly higher levels of TLR4 and produce more pro-inflammatory chemokines.¹¹

In line with the research by Devarapu and Anders, this study showed that there were significant differences in TLR4 expression among the LN, SLE and healthy control groups (p=0.000), with higher TLR4 expression found in the SLE group compared with healthy controls, and the highest expression found in the LN group. This finding suggests that TLR4 may be involved in the development of LN. This result is also in accordance with the research conducted by Wu et al, which showed that although TLR4 gene polymorphisms (Asp299Gly and Thr399 Ile) have no effect on the predisposition and clinical characteristics of SLE, the level of TLR4 messenger RNA (mRNA) expression in peripheral blood mononuclear cells of patients with SLE is much higher than in healthy controls.⁷ The study by Summers et al showed that TLR4−/− mice demonstrated a global decrease in cytokine and autoantibody production. Renal injury was attenuated in TLR4−/− mice, which demonstrated less glomerular immunoglobulin and complement deposition. These results demonstrate that both TLR9 and TLR4 are required for ‘full-blown’ autoimmunity and organ injury in experimental lupus induced by pristane.¹⁷

TLR4 has the most complex signalling pathway of all TLRs, consisting of two main pathways. The first is the MYD88-TIR Domain-Containing Adaptor Protein (TIRAP) pathway. The MyD88-TIRAP pathway regulates the activation of NF-κB, a family of transcription factors that can induce the expression of various pro-inflammatory genes, including those encoding cytokines and chemokines, and also participate in the regulation of inflammation. In addition, NF-κB plays a critical role in regulating the survival, activation and differentiation of innate immune cells and inflammatory T cells. Dysregulation of NF-κB activation contributes to the pathogenic process of various inflammatory diseases.¹⁸ The DNA-binding protein HMGB1 and lupus autoantigen, released under inflammatory conditions, can induce NF-κB activation in a TLR4-receptor and advanced glycation end product (RAGE)-dependent manner in mononuclear phagocytes, neutrophils and mesangial cells.¹¹ Patients with LN exhibit increased expression and activation of NF-κB in glomerular and mesangial endothelial cells, accompanied by increased levels of inflammatory cytokines. NF-κB activation is also responsible for the production of inflammatory mediators, such as reactive oxygen species (ROS) and inducible nitric-oxide synthase (iNOS), which aggravate the symptoms of the disease.¹⁹ The selective IKK inhibitor Bay11-7082 improves the LN mouse model by inhibiting NF-κB and NLRP3. Consistently, genes encoding negative regulators of NF-κB, namely A20 (tumour necrosis factor (TNF)-alpha-induced protein 3) and A20 binding inhibitor (TNF-alpha-induced protein 3-interacting protein 1), have been associated with SLE and LN in humans. A20 is a ubiquitin-editing enzyme, while A20 binding inhibitor of NF-κB1 (ABIN1) is a ubiquitin-binding protein that inhibits IKK/NF-κB signalling. A20 deficiency is associated with autoimmune and inflammatory diseases, including lupus. Genetic studies in both humans and mice have also suggested the involvement of ABIN1 in the development of autoimmune nephritis. ABIN1-deficient mice display aberrant NF-κB activation and develop lupus-like autoimmunity, along with pathological symptoms resembling LN.

Li et al found that, after TLR4 recognition of extracellular HMGB1, AP-1 and IRF3/5/7 are upregulated, promoting the transfer of NF-κB to regulate the expression of genes encoding inflammatory cytokines through the TLR4 adapter MyD88 and TRIF. Additionally, NF-κB activity is essential for immune cell activation.^{8 20} A study by Kirchner et al showed increased levels of HMGB1, TLR4, NF-κB and their downstream target genes TNF-α and interleukin (IL)-1β in MRL/lpr mice, compared with control mice, along with more severe inflammatory damage in kidney tissues. These results highlight the strong inflammation-mediating role of the HMGB1/TLR4/NF-κB pathway in LN.²¹ Yu et al also found that the levels of TLR4 and MyD88 expression, as well as the nuclear translocation of NF-κB, were significantly increased in glomerular endothelial cells of MRL/lpr mice and human renal glomerular endothelial cells affected by LN.²² All of these studies were in accordance with this study, which showed significant differences in NF-κB expression between the LN, SLE and healthy control group. Higher NF-κB expression was found in the SLE group compared with healthy controls, with the highest expression observed in the LN group. There was also a significant correlation between TLR4 and NF-κB, with a positive correlation and a strong association. Linear regression analysis between TLR4 and NF-κB showed that the effect of TLR4 on NF-κB was 81.7%, suggesting that NF-κB may be involved in LN development through the role of TLR4.

The second TLR4 signalling pathway involves the TIR-domain-containing adaptor inducing interferon-β (TRIF) and the TRIF-related adaptor molecule (TRAM). The TRIF-TRAM pathway activates the transcription factor IRF3 and induces the production of type 1 IFNs, namely IFN-α and IFN-β.¹² IRF3 is a transcriptional regulator of cellular responses in various cell types and plays a crucial role in innate immunity.^{13 14} IRF3 is constitutively expressed in various cell types, where it remains inactive in the cytoplasm. On activation of pattern recognition receptors by pathogen infection or synthetic ligand stimulation, IRF3 undergoes phosphorylation and translocates into the nucleus, where it then induces the transcription of type I IFN genes. In addition to type I IFN genes, IRF3 can directly target several cytokine genes, including CXCL10, RANTES, IFN-stimulated 56, IL-12p35, IL-15 and arginase II. On the other hand, IRF3 also functions as a negative regulator of gene expression. For example, IRF3 is directly recruited to the promoter and enhancer of the Il12b gene in response to RIG-I-like receptor (RLR) activation and suppresses Il12b mRNA through competition with IRF5. Furthermore, IRF3, activated by RLR signalling, interacts with the transcription factor SMAD3 to suppress the transforming growth factor-β response in the innate immune response.¹⁴ A study showed that the IRF3 gene variant rs2304206 may be associated with SLE, based on a cohort study of 156 patients with SLE of Mexican Mestizo descent and 272 controls. However, another study failed to confirm the genetic association of IRF3 (rs7251, rs2304204 and rs2304207) with SLE in a Spanish population of 610 patients with SLE and 730 healthy controls.^{13 23} The transcription factor IRF3, activated through the TRAM-TRIF pathway in TLR4 signalling, plays an important role in the induction of type I IFNs, IFN-α and IFN-β, which occurs on activation of signal-transducing pattern recognition receptor (PRR)s of the innate immune system. IRF3 may also be involved in unfavourable immune responses, such as in pro-inflammatory conditions and autoimmune diseases.¹⁴ A study by Zhang et al found that IRF3 rs7251 was specifically associated with LN. IRF3 is likely involved in the pathogenesis of SLE through T-cell differentiation, possibly by repressing T-reg effector lymphocytes.¹³ In line with previous research, this study showed significant differences in IRF3 expression between the LN, SLE and healthy control groups. Higher IRF3 expression was found in the SLE group compared with healthy controls, with the highest expression observed in the LN group. There was a significant correlation between TLR4 and IRF3, with a positive correlation and strong association. Linear regression analysis between TLR4 and IRF3 showed that the effect of TLR4 on IRF3 was 89.6%, suggesting that IRF3 may be involved in LN development through the role of TLR4. These results indicate the strong inflammation-mediating role of the TLR4/IRF3 pathway in LN.

Although the diversity in illness progression and response to pristane could be confounding factors in this study, it can be concluded that TLR4, NF-κB and IRF3 may be responsible for the development of nephritis in SLE. The increased expression of NF-κB and IRF3 likely occurs through the activation of TLR4. Increased expression of these biomarkers in lupus without nephritis may indicate progression towards nephritis, which still needs to be proven with further research.

The authors thank Troef Soemarno, MD (Department of Anatomical Pathology, Universitas Hang Tuah, Surabaya, Indonesia) and Anny Setijo Rahaju, MD (Department of Anatomical Pathology, Universitas Airlangga, Surabaya, Indonesia), for their expertise in evaluating the renal biopsy specimens and all those involved in this research.

Data availability statement

Data are available in a public, open access repository.

Ethics statements

Patient consent for publication

Not applicable.

Ethics approval

The study was approved by the Health Research Ethics Committee, Faculty of Medicine, Brawijaya University (No. 145/EC/KEPK-S3/07/2023), after an ethical review based on the Declaration of Helsinki.

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