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.

Keywords: autoimmune diseases; inflammation; lupus erythematosus, systemic; lupus nephritis.

Figure 1. Histopathology of mice kidney tissue. (A) Normal glomerulus in H&E stain, (B) normal glomerulus in PAS stain, (C) normal glomerulus in MT stain, (D) normal glomerulus in JMS stain, (E) mesangial hypercellularity in H&E stain (red arrow), (F) cellular crescent in PAS stain (red arrow), (G) focal segmental glomerulosclerosis demonstrate blue collagen deposition in MT stain (red arrow), (H) immune deposition at the basement membrane in JMS stain (red arrow). JMS, Jonas Methenamine Silver; MT, Masson’s trichrome; PAS, Periodic Acid-Schiff.

Figure 2. TLR4, NF-κB and IRF3 expressions in kidney tissue. (A) TLR4 expressions in healthy control group, (B) TLR4 expressions in SLE group, (C) TLR4 expressions in LN group, (D) NF-κB expressions in healthy control group, (E) NF-κB expressions in SLE group, (F) NF-κB expressions in LN group, (G) IRF3 expressions in healthy control group, (H) IRF3 expressions in SLE group, (I) IRF3 expressions in LN group. IRF3, interferon regulatory transcription factor 3; LN, lupus nephritis; NF-κB, nuclear factor kappa B; TLR4, toll-like receptor 4.

Figure 3. Correlation of TLR4 with NF-κB and IRF3. (A) Correlation between TLR4 and NF-κB. (B) Correlation between TLR4 and IRF3. IRF3, interferon regulatory transcription factor 3; NF-κB, nuclear factor kappa B; TLR4, toll-like receptor 4.