Assessment of the role of high-density lipoproteins and their immunomodulatory activity in systemic lupus erythematosus immunopathology

Introduction

Systemic lupus erythematosus (SLE) is a complex autoimmune disorder with a wide and diverse spectrum of clinical manifestations and unclear aetiology, although it markedly affects young women of fertile age. Its clinical assessment and management depend on the reckoning of disease activity, organ damage and ailing quality of life. The epidemiology of SLE is inconsistent, and most countries, especially non-developed countries, have limited data.1 According to the global burden of this disease,1 the incidence ranges from 1.4 to 15.13 per 100 000 person-years, and the prevalence ranges from 13 to 7713 per 100 000 individuals, with higher epidemiological rates for women. SLE immunopathology begins with exposure to autoantigens, exacerbated by inflammation from infections, trauma or prolonged ultraviolet radiation exposure. In addition, dysfunctional immune regulation leads to self-molecule recognition, driving an autoimmune response mainly through type I interferon (IFN), particularly IFN-α, produced by plasmacytoid dendritic cells. This IFN-α promotes T-cell proliferation, B-cell activation and inflammation.2 Pathogenic autoantibodies are subsequently produced and circulate in the blood, where they target self-molecules in various tissues and form immunocomplexes that lead to chronic inflammation.3 The importance of inflammation in SLE is clear, and in this regard, studying the mechanisms underlying this inflammation would provide a solid understanding of the immunopathology of this disease, providing interesting therapeutic approaches.4 Disease activity assessment in patients with SLE is crucial for identifying where and how the immune system is active, thus allowing better patient management.5 For this purpose, validated indexes that combine clinical and laboratory data are employed. The most commonly used SLE Disease Activity Index (SLEDAI) has different variations.6 It has been shown that proinflammatory cytokines induced by either type I IFNs or immunocomplexes such as interleukins IL-1β, IL-6, IL-15, IL-18 and tumour necrosis factor alpha (TNF-α) are effectively associated with disease activity and thus indicate SLE-derived inflammation.7 Inflammasomes are associated with inflammation via IL-1β and IL-18 maturation and release. Inflammasomes are large multiproteic complexes composed of oligomerised intracellular sensors of the nucleotide oligomerisation domain (NOD-like) receptor family, adaptor proteins and pro-caspase-1, which, once cleaved into active caspase-1, can process pro-IL-1β and pro-IL-18 into their active forms.8 Research has shown the upregulation of inflammasome mRNA expression and activity in human and murine models of SLE, suggesting potential implications for chronic inflammation.9 10 Although inflammasomes do not cause SLE, they play a role in the immunity impairment in SLE and thus represent an interesting therapeutic target. On this basis, identifying novel molecules contributing to therapeutic strategies related to inflammasome modulation could provide a more useful approach to the regulation of this inflammatory mechanism. One of these candidates is high-density lipoproteins (HDL). HDL are plasma macromolecular complexes related to lipid metabolism and are composed of lipids, apoproteins and associated proteins, whose primary function is the reverse transport of excess cholesterol from peripheral tissues to the liver before excretion.11 Recently, these HDLs have been implicated in immunological regulation via anti-inflammatory mechanisms. HDL can bind bacterial products and endotoxins, reducing inflammatory responses. HDL can also decrease the expression of adhesion molecules such as selectins and the integrin ligands VCAM-1 (vascular cell adhesion molecule 1) and ICAM-1 (intercellular adhesion molecule 1), consequently reducing immune cell tissue infiltration.12 They also participate in lipid raft composition, which can negatively regulate proinflammatory cytokine signalling pathways.13 HDL has also been shown to participate in apoptosis homeostasis by promoting apoptotic body and cell debris clearance.14 Furthermore, HDL can negatively modulate the activation of inflammasomes.15 Nevertheless, most studies on HDL in patients with SLE have focused almost exclusively on cardiovascular risk.16 17 Therefore, there is an interesting scope around the immunomodulatory capabilities of HDL in the context of systematic lupus erythematosus and its relation to inflammation. Moreover, it is well known that HDLs are quantitively and qualitatively altered in lupus patients.18 In this sense, this study aimed to assess the associations among HDL levels, the expression of inflammasome components and other inflammatory markers in the context of SLE immunopathology.

Methods

Results

Patients with SLE have decreased HDL cholesterol levels in contrast to healthy individuals

We quantified HDL-C and detected lower levels in this group of patients with SLE than in healthy individuals with similar features in terms of age, sex and some lifestyle habits (p<0.05, figure 1A). We also observed that lower HDL-C seems to be dependent on disease activity, as patients classified with active lupus (SLEDAI-2K score ≥4) had even lower levels than both healthy controls and inactive patients with SLE (p<0.001 and p<0.05, respectively; figure 1B). We also assessed whether cholesterol control or immunosuppressive medication could alter HDL-C in patients with lupus. We found that HDL-C levels were not significantly different in patients taking cholesterol-reducing statins (figure 1C). However, when patients were taking corticosteroids, their HDL-C levels were lower than those of healthy controls (p≤0.05). In contrast, the HDL-C levels of patients who were not receiving corticosteroid therapy were not significantly different from those of healthy controls (figure 1D).

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Figure 1

Serum HDL cholesterol levels in individuals with systemic lupus erythematosus (SLE) and healthy controls. (A) Comparison of HDL levels between patients with SLE and healthy controls. (B) Comparison of HDL levels between healthy controls and active or remission patients with SLE. Additionally, the HDL levels of controls and patients with SLE were compared according to the use of statins (C) and corticosteroids (prednisone or methyl-prednisone) (D) in the SLE group. The dotted lines indicate a normal HDL level when estimating cardiometabolic risk. The statistical comparison was made using Mann-Whitney U and Kruskal-Wallis tests with a 95% confidence level, and when applicable, post hoc tests (or multiple benchmarks) for the HDS of Dunn were conducted. Statistically significant differences are represented as asterisks for every pair of groups compared (*p<0.05; **p<0.01; ***p<0.001; and ****p<0.0001). Control n=50, SLE n=50, SLEDAI<4 n=28, SLEDAI≥4 n=22, statin use n=10, no statin use n=40, corticosteroid n=29, no corticosteroid n=21. HDL, high-density lipoprotein; HDL-C, HDL cholesterol; HDS, honestly significant difference; SLEDAI, SLE Disease Activity Index.

Compared with healthy individuals, patients with SLE have increased proinflammatory cytokine levels

As expected, individuals with SLE carry an inflammatory burden that is partly mediated by proinflammatory cytokines; the levels of the cytokines IL-6 and IL-18 are usually increased in these patients and are correlated with disease activity,21 22 yet the serum IL-1β level in patients with SLE is still unclear.23 We measured the cytokine levels in the serum and found that the levels of both cytokines were significantly greater in the SLE population (p<0.0001, figure 2A, C, online supplemental table S2). We also observed that the increased levels of both IL-6 and IL-1β were not significantly different between patients with active and remission SLE (figure 2B, D). No significant differences in CRP levels were detected between patients and controls (figure 2E, F).

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Figure 2

Serum inflammatory markers in individuals with systemic lupus erythematosus (SLE) and healthy controls. Comparison between (A) IL-6, (C) IL-1β and (E) CRP serum levels in patients with SLE and healthy controls. Additionally, patients with SLE were grouped according to disease activity in (B), (D) and (F). Dotted lines indicate a normal CRP level when estimating cardiometabolic risk. The statistical comparison was established using Mann-Whitney U and Kruskal-Wallis tests with a 95% confidence level, and when applicable, post hoc tests (or multiple benchmarks) for the HDS of Dunn were applied. Statistically significant differences are represented as asterisks for every pair of groups compared (*p<0.05; **p<0.01; ***p<0.001; and ****p<0.0001). Control n=50, SLE n=50, SLEDAI<4 n=28, and SLEDAI≥4 n=22. HDS, honestly significant difference; hsCRP, high-sensitivity C reactive protein; SLEDAI, SLE Disease Activity Index.

Gene expression of inflammasome-related genes is upregulated in the PBMCs of individuals with SLE compared with healthy controls

The assessment of inflammasome-related gene expression in patients with SLE is still somewhat lacking. However, there is substantial evidence of the upregulation of NLR receptors gene expression in lupus patients.24 Nevertheless, inflammasomes are generally acknowledged to participate directly in SLE immunopathology. Our results support this idea, as we found that the gene expression of both IL-1β and IL-18 was positively regulated together with that of all inflammasome components related to canonical activation (figure 3A–D, online supplemental table S2). Similarly, the gene expression of the receptors NLRP1, NLRP3, NLRC4 and AIM2 was also enhanced in patients with SLE (figure 3E–H). This was expected for NLRP3 and AIM2, in particular, as increased gene expression of these receptors has been associated with SLE.25 We also investigated whether these gene expression levels were dependent on disease activity and found that only the mRNA expression of IL-1β was upregulated in patients with active SLE (SLEDAI-2K score ≥4) but not in patients in remission (online supplemental figure S1). The other gene expression markers evaluated showed no differences between patients with active SLE and patients in remission (online supplemental figure S1).

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Figure 3

Gene expression of inflammatory markers related to inflammasomes in individuals with systemic lupus erythematosus (SLE) and healthy controls. Comparison of relative gene expression between patients with SLE and healthy controls. IL-1β (A), Caspase-1 (B), IL-18 (C), ASC (D), NLRP3 (E), NLRP1 (F), NLRC4 (G) and AIM2 (H). PGK was used as the constitutive gene to normalise expression levels. The statistical comparisons were conducted using the Mann-Whitney U test. Statistically significant differences are represented as asterisks for every pair of groups compared (*p<0.05; **p<0.01; ***p<0.001; and ****p<0.0001). AIM2, absent in melanoma 2; ASC, apoptosis-associated speck-like protein; NLRC4, NOD-like receptor CARD domain containing 4; NLRP1, NOD-like receptor Pyrin domain containing 1; NLRP3, NOD-like receptor Pyrin domain containing 3; PGK, phosphoglycerate kinase; RTU, relative transcription units.

HDL-C levels in patients with SLE are associated with inflammasome component gene expression and serum proinflammatory cytokines

According to the differences, we observed in both groups regarding all markers assessed and HDL levels, we investigated possible associations between HDL levels and inflammatory markers in patients with SLE. Negative correlations were found between HDL and disease activity (figure 4A), between the serum concentrations of the cytokines IL-6 and IL-1β (figure 4B, C), or between the gene expression of the inflammasome canonical activation elements NLRP3, IL-1β and IL-18 (figure 4D–F). Additionally, as we found disease activity to be associated with HDL, we explored whether this activity was related to any of the markers measured. Positive correlations were observed between disease activity and serum IL-1β concentration (figure 5A) and between disease activity and IL-18 gene expression (figure 5B). Following these results, we examined this association by segregating disease activity in the active and remission lupus groups. Surprisingly, we found that active lupus (SLEDAI-2K score ≥4) correlated with serum IL-1β (figure 5C) and with NLRP3 (figure 5D), even though NLRP3 did not correlate with the SLEDAI ungrouped (p=0.09, r=0.24). (online supplemental figure S2).

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Figure 4

High-density lipoprotein (HDL) levels and the gene expression of inflammasome components and inflammatory markers in individuals with systemic lupus erythematosus are negatively correlated. The correlation between HDL and (A) disease activity (r: −0.4229. P: 0.0022), (B) serum IL-6 (r: −0.2895. P: 0.0415), (C) serum IL-1β (r: −0.3093. P: 0.0288), (D) NLRP3 (r: −0.6053. P: <0.0001), (E) IL-1β (r: −0.3320. P: 0.0211), (F) IL-18 (r: −0.4119. P: 0.0036) mRNA expression. The dotted lines indicate a normal HDL level when estimating cardiometabolic risk. Correlations were assessed with Spearman’s Rho test. P <0.05 were considered significant. HDL-C, HDL cholesterol; NLRP3, NOD-like receptor Pyrin domain containing 3; RTU, relative transcription units SLEDAI-2K, SLE Disease Activity Index 2000.

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Figure 5

Disease activity and the gene expression of inflammasome components and inflammatory markers in individuals with systemic lupus erythematosus (SLE) are positively correlated. Correlation between the SLEDAI-2K score and (A) serum IL-1β (r=0.4828. P: 0.0004), (B) IL-18 (r: 0.3785. P: 0.0080) mRNA expression. Correlations between active SLE (SLEDAI-2K score≥4) and (C) serum IL-1β (r=0.5168. P: 0.0138), (D) NLRP3 (r: 0.4999. P: 0.0179) mRNA expression. The dotted lines indicate the active SLE threshold according to the SLEDAI-2K score. Correlations were assessed with Spearman’s Rho test. P <0.05 were considered significant. NLRP3, NOD-like receptor Pyrin domain containing 3; RTU relative transcription units; SLEDAI-2K, SLE Disease Activity Index 2000.

Discussion

Chronic inflammation in SLE is a hallmark of the disease and a common feature among most patients4; many mechanisms derived from autoimmunity and lack of immunoregulation contribute to this inflammation. Immune complexes (ICs), which are a direct consequence of recognising self-molecules, are one of the main sources of this immune dysregulation.26 Following deposition in a tissue endothelium, ICs trigger inflammation in different ways: first, they activate the complement system, which leads to tissue damage and cellular death; this releases nuclear material, such as nucleic acids, and generates cell debris as a source of autoantigens.27 Then, ICs formed by Acs bound to nucleic acids and their associated proteins lead to the opsonisation of those autoantigens. In addition, several immune regulatory mechanisms are usually dysregulated, such as cell apoptosis, necrosis and neutrophil extracellular trap (NET) clearance, which keep these antigens exposed. Once self-antigens are bound by Acs in ICs, they can be recognised and internalised by the Fc receptors (FcRs) of several immune cells, including dendritic cells (conventional, follicular and plasmacytoid), macrophages, neutrophils and some lymphocytes.28 Internalised ICs stimulate intracellular patter-recognition receptors (PRRs) such as endosomal Toll-like receptors (TLRs) or cytosolic NLRs. Depending on the cell, different effects can be triggered: in follicular dendritic cells, ICs promote antigen presentation, interaction with B cells and, therefore, increase affinity maturation and class switching29; in plasmacytoid dendritic cells (DCs), ICs stimulate the excessive production of IFNα, which promotes autoimmunity and inflammation through various mechanisms30; and in neutrophils, ICs can favour NETosis, which is a type of cellular death that ends with the release of chromatin from neutrophil chromatin that forms fibres with antimicrobial peptides and enzymes known as NETs that can also attach autoantigens.31 In the case of lymphocytes, ICs have been shown to increase the capacity of B and T cells to respond to antigen stimulation.32 ICs stimulate macrophages and monocytes to produce and secrete proinflammatory mediators such as MCP-1 and TNF-α, among others, as well as clearance of ICs by macrophages.26

To some extent, these inflammatory effects are related to inflammasomes and their associated molecules.4 In this respect, studying inflammasomes and their activation in this inflammatory environment have proven quite interesting. Globally, it is acknowledged that inflammasomes, mainly NLRP3, contribute to SLE pathogenesis, as it has been observed that these multiprotein complexes are upregulated and hyperactive in patients with SLE.24 This upregulation has been primarily found in PBMCs and macrophages, and NLPR3 gene expression is increased in renal epithelial cells. Furthermore, this overexpression and hyperactivation are positively associated with disease activity and organ damage.33

Interestingly, it has been reported that double-stranded DNA and autoantibodies against this molecule can activate the inflammasome NLRP3, increasing signalling in the NF-kB pathway and caspase-1 expression with enhanced IL-1β and macrophage migration inhibitory factor production in human PBMC monocytes.25 An exciting study by Kahlenberg et al 9 showed that NETs derived from patients with SLE could activate inflammasomes, promoting the secretion of IL-1β and IL-18, similar to the antibacterial peptide IL-37, in murine and human macrophages.9 Moreover, type I IFNs have been shown to stimulate inflammasome-related gene expression in patients with SLE monocytes via IFN regulatory factor 1.34

In this study, and in agreement with these studies, PBMCs from patients with SLE exhibited increased expression of inflammasome components and their products, mainly NLRP3, suggesting an increased inflammatory status related to this mechanism. The serum concentration of IL-1β was also increased, although there is no consensus on the serum concentration of IL-1β in patients with SLE. This could be explained by differences in the sensitivity of the measuring assays used; nonetheless, upregulation of IL-1β mRNA expression is well described and characterised,10 35 indicating a logical biological connection in our results. Additionally, we found a positive correlation between NLRP3 and IL-1β and disease activity, which further strengthens the evidence of the pathogenic role of this mechanism in SLE-related inflammation.

However, assessments of other inflammasomes, such as NLRP1, NLRC4 and AIM2, are lacking, and their involvement in SLE immunopathogenesis is unclear. In the cases of NLRP1 and NLRC4, some genetic polymorphisms have been associated with increased SLE risk.36 AIM2 has been reported to exhibit dual behaviour, since some reports have shown that high levels of AIM2 correlate with inflammatory status and disease progression in patients with SLE and mice and that AIM2 silencing by small interfering RNA knockdown reduces disease severity and inflammation.37 In contrast, other studies have shown that negative regulation of AIM2 can also promote lupus pathogenesis in mice by increasing the IFN response mediated by the IFN-inducible p202 protein.38 Our results revealed that the gene expression of three NOD-like receptors was upregulated but not associated with disease activity. This indicates that they could be related to the inflammatory status of patients to some extent, as their activation ultimately leads to the production of proinflammatory cytokines; hence, their involvement in other mechanisms and regulation of other pathways remains to be further explored.

Inflammasomes play a crucial role in SLE pathogenesis; this fact has made this inflammatory mechanism an attractive therapeutic target. It has been shown in murine models of lupus that the inhibition or regulation of NLRP3, caspase-1 or ASC can delay and ameliorate SLE development and progression.39 On the other hand, decreased HDL-C levels are common in patients with chronic inflammation. Patients with SLE show an altered lipid profile known as the lupus dyslipoproteinemia pattern, whereas very low density lipoprotein (VLDL) and triglyceride levels are increased, and HDL levels are decreased.40 In this sense, HDL, an immunomodulator related to inflammasome activity, represents a novel and attractive complementary therapeutic target. It has been reported that HDL in vitro can dampen NLRP3 activation and IL-1β production in response to the phagocytosis of cholesterol crystals, a potent inflammasome inductor, by monocytes.11 12 Moreover, data suggest that HDL can also downregulate the expression of inflammasome components in the absence of stimuli, possibly through cross-talk between intracellular signalling pathways.41 Furthermore, serum amyloid A (SAA), an apolipoprotein produced as an acute-phase reactant in response to inflammatory stimuli and with potent proinflammatory activity, can activate the NLRP3 inflammasome and promote IL-1β secretion.42 HDL is associated with SAA and diminishes its capacity to stimulate inflammasome activation.43 Oxidised low-density lipoprotein is a lipid transport molecule closely related to atherosclerosis and inflammation. Additionally, they can act as the two stimuli necessary for two-phase inflammasome activation. Furthermore, they can be recognised by antibodies when in circulation, generating ICs.44 HDL modulates and prevents the oxidation of LDL via paraoxonase-1, thereby reducing the inflammatory effects of LDL and indirectly modulating inflammasome activation.45

On this basis, considering that our patients with SLE had lower HDL levels that are correlated with active SLE, our results suggest that HDL alterations, at least quantitatively, can influence inflammasome expression and activation in patients with SLE. First, HDL levels in our SLE cohort were reduced in proportion to disease activity, which is consistent with what has been reported by other researchers.46–48 This reduction in HDL levels is associated with increased expression of the inflammasome canonical NLRP3 inflammasome pathway at the gene and protein levels; this finding is remarkable, as it suggests that HDL potentially modulates inflammasome activation in these patients with SLE and thus could represent a novel approach for future therapies.

Although the functions of CRP in SLE are complex, moderate elevations in CRP levels are usually found in patients with SLE, but these elevations are not associated with disease activity or the extent or severity of inflammation. However, it has been proposed that CRP could promote cellular debris and IC clearance via FcRs, as well as via classical complement activation in SLE, and could also modulate IFN-α responses derived from IC processing in plasmacitoid dendritic cells (pDCs).49 We wanted to evaluate whether CRP could also participate in inflammation and inflammasome regulation, but we did not observe differences in CRP (figure 2) levels or any correlation between CRP and the inflammatory markers we evaluated (online supplemental figure S2).

On the other hand, comorbidities would likely not explain the general finding of inflammation observed in all the patients, as only 2 of them had diabetes mellitus, 7 had osteoporosis and 13 had arterial hypertension, which represents a minority of the population we evaluated. Additionally, IL-6 is a versatile cytokine with pleiotropic functions, including proinflammatory and anti-inflammatory effects. SLE is associated with pathological inflammatory responses and disease activity.22 Our results revealed that IL-6 was also increased in the SLE group, indicating a possible inflammatory role associated with the disease rather than other causes.

Finally, our study had some limitations. First, the lack of evaluation of other HDL dimensions such as their functionality and fraction composition. Second, we did not exclusive selected patients with active SLE, which could have limited a more precise association between disease activity and inflammasome activation markers. Third, disease activity measured through SLEDAI-2K was not independently assessed by more than one assessor. Finally, given the characteristics of the study, we could not approach experimentally any specific cellular or molecular mechanisms that could explain our results better. However, to our knowledge, this is the first study to simultaneously assess HDL and inflammasome expression and activation and their association with disease in a Colombian cohort of patients with SLE.

Conclusions

Our results suggest that HDL plays an essential role in SLE beyond atherosclerosis, given that this molecule can immunomodulate the inflammatory environment seen in these patients, which is one possible mechanism for this regulation of inflammasome expression and activation. This opens new possibilities for the treatment, management and study of SLE. In this sense, further lipidomic and transcriptomic studies, as well as mouse lupus animal models focused on the immunomodulatory capabilities of HDL, will likely generate helpful knowledge and insights into lipid metabolism and immune regulation dynamics in patients with lupus and autoimmune diseases.