Systemic lupus erythematosus (SLE) is a disorder of immune regulation resulting in chronic inflammation that affects many organs. Treatment of lupus disease must be determined individually, since different patients may have diverse and multiple symptoms with variable severity. Mild disease requires no or little therapy, usually nonsteroidal anti-inflammatory medications (NSAIDS). Articular and skin symptoms are frequently treated with antimalarial drugs, especially hydroxychloroquine, alone or with low dose corticosteroids when required, whereas severe lupus must be treated with corticosteroids and/or immunosuppressive drugs. However, it is assumed that responses to specific agents may be variable among SLE patients. Consequently, the identification of genetic predictors of treatment response would provide valuable clinical information, since these can be determined at the time of diagnosis, when therapeutic intervention has the potential to offer the greatest benefits.

Antimalarial drugs (hydroxychloroquine, chloroquine and quinacrine)have been used as disease-modifying antirheumatic agents in the treatment of several autoimmune diseases, usually associated with increased secretion of pro-inflammatory cytokines. However, despite an extensive clinical history of use in rheumatoid arthritis and SLE 1, their mechanisms of anti-inflammatory action have not yet been fully defined. Chloroquine is thought to concentrate in acidic subcellular compartments, such as endolysosomes, where it inhibits acidic proteases 2. As lysosomal enzymes are involved in antigen processing and presentation, an antirheumatic effect might be mediated by a downregulation of the immune response against autoantigens. However, the effects of chloroquine and related drugs may extend beyond this. Interestingly, antimalarials have been shown to inhibit the release of the pro-inflammatory cytokines IL-1, IL-6 and tumor necrosis factor (TNF)α by monocytes activated with lipopolysaccharide (LPS) or CpG oligonucleotides 3456 and it has been reported that chloroquine interferes with LPS-induced expression of the gene encoding TNFα in human blood monocytes by a nonlysosomotropic mechanism 7. However, it is not yet known whether treatment of humans with antimalarial drugs is capable of reducing pro-inflammatory cytokines in vivo.

Considering the central role that IL-10 and TNFα cytokines play in the pathogenesis of SLE, it is possible that different cytokine production may not only affect the natural course of the disease, but also the response to therapy. Genetic polymorphisms at the promoter of the genes encoding IL-10 and TNFα have been associated with different constitutive and induced cytokine production. The genetic variant at position -308 (G/A) of the gene encoding TNFα was found to have functional effects on gene transcriptional activity, carriers of the uncommon TNF2 allele (-308A*) being considered as genetically high TNFα producers 8910. Similarly, IL-10 basal and induced production presented interindividual variations that were genetically regulated by three single nucleotide polymorphisms (SNPs) at positions -1,082(G/A), -819(C/T) and -592(C/A) of the IL-10 promoter. In Caucasian populations, only three haplotypes have been found (GCC, ACC and ATA), the individuals GCC/GCC being considered as genetically high IL-10 producers 101112. Several studies have analyzed the association of IL-10 or TNFα genetic variants with susceptibility to and outcome of SLE and other autoimmune diseases, showing variable results in most cases. However, it is known that the actions of cytokines may be profoundly conditioned by the presence of other cytokines, this being particularly true in the case of IL-10 and TNFα, which are mutually regulated and have complex and predominantly opposing roles in systemic inflammatory responses. In a previous study, we found that carriers of the combined genotype high TNFα/low IL-10 have the highest risk factor for developing SLE and producing antibodies to the SSa antigen 10, suggesting that interactions between IL-10 and TNFα cytokine genes may influence susceptibility to SLE, its phenotype and possibly the clinical response to disease modifying antirheumatic drugs. The aim of the present study was to detect the effect of SLE treatments on TNFα serum levels and to evaluate the possible influence of IL-10 and TNFα functional genetic polymorphisms on the response to antimalarial drugs.

In this study we show that SLE patients receiving single or combined treatment with antimalarial drugs have TNFα serum levels similar to healthy controls, whereas untreated patients and those receiving corticosteroid or immunosuppressive therapies presented increased amounts of this cytokine. These results suggest a very valuable effect of antimalarial treatment by means of the downregulation of in vivo TNFα levels. Although these pharmacological agents have been widely used as disease-modifying antirheumatic drugs mainly in the treatment of SLE and rheumatoid arthritis 1, their mechanisms of anti-inflammatory actions have not yet been completely understood. Several in vitro experiments have demonstrated that quinacrine and related drugs decreased the release of pro-inflammatory cytokines induced by LPS in macrophages 456715. In mouse models, it has been reported that chloroquine may protect mice from lethal challenge by CpG oligonucleotides and LPS and may decrease serum TNFα and IL-6 in rats injected with sublethal doses of both stimuli 16. To the best of our knowledge, however, this is the first report demonstrating an in vivo association between the use of antimalarial therapy and low levels of serum TNFα, suggesting that the disease-modifying antirheumatic effect of these drugs may be mediated, at least in part, by a strong downregulatory effect on TNFα production.

However, association between antimalarial treatment and TNFα serum levels seems to be influenced by polymorphisms of the genes encoding TNFα and IL-10, indicating that this advantageous connection may only be completely valuable for patients with a specific genotype. Constitutive and induced TNFα and IL-10 production have important interindividual variations that are genetically regulated by SNP at their promoters 89101112. Our data indicate that antimalarial therapy plays a role in the TNFα production of patients who are genetically high TNFα producers. These patients probably have the highest TNFα transcription rates 9 and consequently the highest serum levels. Although the mechanisms of in vitro antimalarial-mediated TNFα inhibition 4567 are not yet entirely known, it has been reported that chloroquine inhibited LPS-induced TNFα transcription 67, interfering with mitogen-activated protein kinase signaling 17. Thus, our results suggest that antimalarial agents require a high rate of TNFα transcription to achieve the maximal inhibitory effect. On the other hand, though the relevance of IL-10 was already known in lupus disease, the influence of genetic polymorphisms at the IL-10 promoter on treatment outcome after the use of antimalarials was surprising. Results indicated an involvement of antimalarial treatment in the amount of serum TNFα in SLE patients with a low IL-10 genotype. The limited TNFα downregulatory effect observed in genetically high IL-10 producing patients might be explained by the regulatory feedback mechanism that controls the production of both cytokines, which would lead to a decrease in the TNFα transcription rate in patients producing elevated amounts of IL-10. Supporting this, it has been reported that high IL-10 levels were associated with less effective clearance of Plasmodium falciparum parasites in patients receiving antimalarial therapy 18.

Our data support the idea that the actions of cytokines are profoundly conditioned by the presence of other cytokines, particularly in the case of IL-10 and TNFα, which have opposing roles in systemic inflammatory responses. Thus, on the basis of our previous results, we evaluated the role of the interaction between IL-10 and TNFα genotypes in regulating the response to antimalarial treatment in SLE patients. A strong association was found between carriage of the combined genotype low IL-10/high TNFα and the use of antimalarials for more than three years without the need for any other specific SLE treatment (good responder patients), although the single analysis of the IL-10 genotype did not show significant results. Moreover, the relationship between this combined genotype and treatment outcome was higher than that obtained after single analysis of the gene encoding TNFα, since high IL-10/high TNFα producers were not overrepresented among good responder patients. Taken in conjunction, these results thus indicate that determination of TNFα and IL-10 alleles at the onset of the disease may help identify more suitable candidates for antimalarial treatment and could be used as a genetic predictor of clinical outcome. We would expect SLE patients who are carriers of the pro-inflammatory genotype low IL-10/high TNFα to develop a mild disease presenting a good course under antimalarial therapy. Most of these patients probably developed SLE due to the effect of environmental factors added to their genetically determined high TNFα levels, which could not be modulated by the low production of IL-10. TNFα is a pro-inflammatory cytokine that has been found at elevated levels in the serum of patients suffering SLE and other autoimmune diseases 1920; it has also been suggested that TNFα genotype influences their susceptibility 102122 and, possibly, their clinical response to treatment. In these patients, the elevated TNFα levels may be involved in diverse pathological mechanisms and, therefore, a clinical benefit is to be expected under a treatment that diminishes TNFα production.

Under the assumption that the elevated TNFα levels found in patients with various chronic inflammatory diseases are deleterious, several anti-TNFα therapies are now available to block the action of TNFα. Actually, TNFα-blockage with antibodies has been of unquestionable clinical benefit to many patients with rheumatoid arthritis, Crohn's disease, psoriasis and, more recently, ankylosing spondilitis 2023242526. However, it has been reported that response to this treatment is also influenced by genetic polymorphisms at FcγRIII 27, HLA-DRB1 28, lymphotoxin-α 29 and TNFα alone 30 or combined with IL-10 31 or lymphotoxin-α 28. In fact, TNFα antagonists seem to be more effective in genetically low TNFα producer patients 30 or with the combined high IL-10/low TNFα genotype 31. Several authors have also proposed the use of TNFα lowering agents in the treatment of lupus disease 203233. Supporting this, an open label study of infliximab in six patients with SLE indicated that TNFα blockage might have a therapeutically beneficial effect, although autoantibodies to double-strained DNA and cardiolipin were increased 34. This trend toward an augmented autoantibody production frequently accompanying this treatment was not reported after the use of antimalarials. Given the therapeutical relevance of these results, new studies need to be designed to evaluate the use of these drugs as TNFα downregulators in the treatment of genetically low IL-10/high TNFα producer patients with SLE and other inflammatory diseases who, in addition, are poor responders to TNFα blockage with antibodies.

The limitations of the work were the lack of a prospective longitudinal study analyzing TNFα levels before and after treatment with antimalarials and other SLE therapies and the absence of standardized validated measures of SLE activity.

Our results demonstrate a relationship between antimalarial treatment and low TNFα serum levels in SLE patients that were influenced by polymorphisms at the IL-10 and TNFα gene promoters. Therefore, our findings may have an important clinical application through the identification of patients who are the most likely to benefit from antimalarial therapy.

CI = confidence interval; ELISA = enzyme-linked immunosorbent assay; IL = interleukin; LPS = lipopolysaccharide; NSAIDS = nonsteroidal anti-inflammatory drugs; OR = odds ratio; PCR = polymerase chain reaction; SLE = systemic lupus erythematosus; SNP = single nucleotide polymorphism; TNF = tumor necrosis factor.

The authors declare that they have no competing interests.

PL performed genetic and immunological assays, data collection and statistical analyses and participated in interpretation of data. JG carried out a detailed review of clinical histories and participated in data collection. LM performed patient selection and participated in data collection. CG participated in the study design, interpretation of data and helped to draft the manuscript. AS conceived the study, participated in its design and coordination, performed genetic assays and statistical analyses and drafted the manuscript.