Understanding the pathophysiology of systemic lupus erythematosus (SLE) remains a challenge, given its multifaceted nature involving genetic, immunological, and environmental factors 21–23. In our study, we sought to shed light on this complex condition by evaluating a cohort of individuals diagnosed with SLE, utilizing the Systemic Lupus Erythematosus Disease Activity Index (SLEDAI) score as a measure of disease activity. Our findings revealed that the SLE group exhibited predominantly active disease, as indicated by an average SLEDAI score of 12 points. Notably, 64% of these individuals presented with classification II, III, and IV nephropathies, with respective proportions of 7%, 26%, and 31% among the total study population. This cohort comprised 50 females and 5 males, with an average disease duration of approximately 5 years. Importantly, we compared our findings with those of a control group lacking SLE, providing valuable insights into the distinctive features of SLE pathogenesis and EBV infection.
The role of circulating TNF-α in inflammation is crucial, as it serves as a catalyst for the induction of various proinflammatory molecules and cytokines 24. However, its involvement in systemic lupus erythematosus (SLE) pathogenesis is complex and somewhat controversial. For instance, studies have reported that TNF-α may contribute to susceptibility to SLE through certain polymorphisms 25, through elevated serum levels 26, or by effects on T lymphocytes highly susceptible to TNF-α 27. Furthermore, dysregulated production of TNF-α and IFN-γ, coupled with aberrant B-cell responses, has been implicated in the immunological dysfunction observed in SLE patients 28. These findings underscore the intricate interplay between TNF-α and the immune dysregulation characteristic of SLE, shedding light on potential targets for therapeutic intervention. Previous studies show upregulation of these cytokines in Europeans 26,29; however, in our study, where our population is different from those studies, we observed that the relative expression of both TNFA and INFG decreased significantly in patients with SLE compared to control individuals 17, However, TNF-α also functions as an inflammatory mediator and inducer of apoptosis 30, while deficient TNF-α production leads to the absence of both germinal centers and follicular dendritic cells 31, which in murine models has been associated with lupus development 32. For example, these results could be explained by various factors such as infections such as hepatitis virus, HHV, retrovirus, parvovirus B19, or EBV 33–37, which have been associated with clinical and immunological manifestations similar to those found in SLE.
We investigated immune alterations in a dual context of SLE and viral infection. For this, we explored the presence of active or latent EBV infection in all the individuals. Previous studies have demonstrated that during EBV infection, the virus decreases IFN-g response 38–41. In our study we also show that IFN-g is down regulated in the SLE group as well as having an active viral infection showed by the presence of anti-EBNA, anti-EA, and anti-VCA IgGs (Table 3). The reduction of IFN-g by EBV would probably dysregulate the already exacerbated immune response in individuals with SLE maintaining an overstimulated immune activity particular to SLE 6. In the control group, both IFNG and TNFA are not reduced and would maintain viral expression to low levels as observed in our study where most of the population show seropositivity, but active infection is reduced significantly in the control group.
We identified a significantly over expression of LMP1 in patients with SLE compared to control subjects. LMP1 is a viral protein implicated in B-cell transformation and viral maintenance 42. Similar to our findings, others have demonstrated that EBV infection in individuals with lupus show a 10- to 100-fold higher expression of LMP1 compared to their control groups 43–45, assessed through the viral load in peripheral blood, the frequency of infected B cells, and the amount of virus in serum. It is also important to highlight that this increase in gene expression seems not to be dependent on immunosuppressive therapy that may be ongoing to treat SLE 43,44,46. These findings becomes relevant since LMP1 is a latent EBV protein with a high potential for altering cellular signal transduction pathways, including blocking intracellular DNA sensors, such as TLR9, and transcription factors, such as IRF3/7 47. These are crucial pathways to promote the proliferation of target cells and, simultaneously, interfere with the regulated processes of apoptosis 48. The influence of LMP1 is exerted through its expression in the plasma membrane, activating signaling pathways, such as NF- κB, protein kinases JNK, and p38 48,49. In SLE patients, an increase in LMP1 could favor an increase in autoreactive B-cell survival, suggesting a mechanism for the higher activity of immune responses seen in SLE patients 50.
Different HERV-E have been identified more frequently in patients with autoimmune disorders, which suggests that the methylation state of the genome contributes to the modulation of the expression of these retrotransposons. Even more so when DNA hypomethylation has been shown to be involved in the pathogenesis of SLE 51. Our results show evidence of a significant relative overexpression of HERV-E gag in patients with SLE, also confirmed by other authors showing increased mRNA expression of HERV-E in CD4 + T cells of patients with lupus 4. Since HERV-E proteins may be structurally similar to autoantigens and trigger autoimmunity through molecular mimicry, potentially serving as a new therapeutic target in lupus 52,53.
There is evidence supporting the role of IL-10 in the promotion of growth and the transformation of auto-reactive B cells into plasma cells in lupus, which, in turn, influences the progress of the disease 54,55. Our results show an increased expression of this cytokine in patients with SLE. Nonetheless, it is worth highlighting that studies on murine models indicate that IL-10 may also play a protective role in lupus as it has proinflammatory and anti-inflammatory effects 56,57. This might also explain the lower expression of TNFA- seen in our studies as IL-10 inhibits TNF-α 58,59. Conversely, the influence of toll-like receptors (TLR) inside the cell, acting as nucleic acid sensors, are an aspect of interest in the pathogenesis of SLE; especially TLR3, which can sense double-stranded RNA (dsRNA) of viral origin, and influence cytokine production by NF-κB signaling pathways 60. This study reports a considerable decrease in the relative expression of TLR3 in patients with SLE; In the context of an active EBV-mediated infection, the expression of non-polyadenylated RNA forming loop structures through base pairing is observed, simulating dsRNA molecules. This phenomenon triggers signaling through TLR3, which is involved in the production of type 1 interferon (type 1 IFN) 61. In our lupus patients, despite exhibiting increased activity of EBV viral infection, their TLR3 expression is significantly diminished, which could imply a factor hindering the immune response to the infection and facilitating viral reactivation. However, it has been demonstrated that in SLE patients infected with hepatitis C virus (HCV), the relative expression levels of TLR3 are higher compared to non-lupus controls also infected 62 reflecting the heterogeneity of these patients' response to infections.
The results of the distribution of the CD45 + pan-leukocyte panel among cell populations in general (neutrophils, monocytes, and lymphocytes) are an important parameter of association with the state of disease activity 12. Conversely, an increase in the expression of CD27 in T and B lymphocytes suggests a crucial role in the immune activation process 63,64. The differences in the distribution of CD27 + B cells are useful for the evaluation of the disease activity in patients with SLE 65, although a low index of disease inactivity also brings about a greater expression of CD27—both for T and B lymphocytes. The different behaviors and possible effects of over and under-expressed genes in the context of lupus are represented in a unified manner based on the results obtained in Fig. 3.
Taken together, these results provide new perspectives to understand the complex immune interactions in SLE and could pave the way for future research and therapeutic approaches. Our study highlights the way in which dysregulation in cytokine activity and the influence of genetic and viral factors, such as EBV, contribute significantly to the heterogeneity of SLE. The variability observed in the expression of TNFA, IFNG, IL-10, and HERV-E gag among patients with SLE not only reflects the diverse immune response, but also stresses the complexity of underlying mechanisms of this disease. These patterns point to a profoundly altered immune system in SLE, where the interplay between intrinsic and external immune factors, such as viral infections, plays a crucial role in pathogenesis. The fact that different patients with SLE show various responses in terms of cytokine and viral protein expression implies the underlying heterogeneity of the disease, which may be vital for customizing the treatment strategies. These findings are the basis for patient stratification and the development of more targeted therapeutic interventions based on the specific biology of each case of SLE.