Interferon-lambda1 induces peripheral blood mononuclear cell-derived chemokines secretion in patients with systemic lupus erythematosus: its correlation with disease activity

Systemic lupus erythematosus (SLE) is an autoimmune and inflammatory disease characterized by the activation of T and polyclonal B lymphocytes, production of numerous autoantibodies, and formation of immune complexes that result in tissue and organ damage [1].

The interferon (IFN) family plays an important role in innate as well as adaptive immune responses against viral infections [2]. Classic IFNs include the type I subgroup composed of IFN-α, IFN-β, IFN-ω, IFN-κ, IFN-τ, and the type II subgroup represented by IFN-γ [3]. Previous studies have suggested that both subgroups play an important role in the pathogenesis of SLE [3‐6].

Type III IFN, IFN-λ1, IFN-λ2, IFN-λ3, also referred to as interleukin (IL)-29, IL-28A and IL-28B, respectively, are novel members of the IFN super-family [7, 8]. They are secreted by human peripheral blood mononuclear cells (PBMC) as well as dendritic cells (DC) upon infection with viruses or stimulation with poly (I:C) or lipopolysaccharide (LPS) [2, 8], and express in a broad spectrum of tissues [7]. Gene expressions are regulated by virus-activated interferon regulatory factor (IRF) 3 and IRF 7 [9]. These proteins induce activation of JAK/STAT signaling pathways through a cell-surface receptor consisting of two chains, IFN-λR1, which is IFN-λ- specific, and IL-10R2, which is shared among IL-10, IL-22 and IL-26 [10, 11].

IFN-λ share several common features with type I IFN, such as antiviral, anti-proliferative as well as antitumor activities [2, 10, 12‐15], meantime, their immune-regulatory function has gradually been elucidated as well. Recent studies have reported that IFN-λ-treated DC specifically induced proliferation of a CD4+CD25+Foxp3+T cell subset [16]. IFN-λ1 was able to inhibit human type 2 helper T (Th2) cell responses by diminishing secretion of IL-13, and also specifically upregulated cytokines IL-6, IL-8 and IL-10 levels secreted by monocytes in a dose-dependent manner [17‐19].

Chemokines are a group of small molecules with the ability to direct cell movements necessary for the initiation of T cell immune response, recruit specific leucocytes to inflammatory sites, regulate polarization of Th1 and Th2 lymphocytes, and influence maturation of DC, T cell and bone marrow progenitor [20‐23]. Moreover, they can stimulate monocytes, natural killer (NK) and T cell migration, and modulate adhesion molecule expansion [24]. Therefore, they are related to tissue inflammation and organ damage in SLE.

IFN-inducible protein-10 (IP-10) is a CXC chemokine secreted by PBMC, fibroblasts and endothelial cells [25], and plays an important role in the perpetuation of chronic inflammatory responses by promoting the recruitment of monocytes, T and NK cells into target tissue and organ [26]. IL-8, another CXC chemokine, is predominantly chemotactic for neutrophils, and also has the capability of recruiting leukocytes to the glomerulus during immune renal damage [27].

Many studies have discovered that plasma chemokine concentrations including IL-8, IP-10 and monokine induced by IFN-γ (MIG) are elevated in patients with active SLE [28‐31]. Some studies have reported that urinary IL-8 levels are increased in SLE patients with active renal disease as well [27, 32].

With this background, we compared expression of IFN-λ1 mRNA in PBMC and serum protein levels in SLE patients with healthy controls. In addition, we determined the correlation of serum IFN-λ1 levels with disease activity and clinical manifestations in SLE, and investigated the effect of IFN-λ1 on the secretion of the chemokines IP-10, MIG and IL-8.

Systemic lupus erythematosus (SLE) is a chronic autoimmune disease affecting multiple organ systems, involving the skin, joints, heart, lungs, kidneys and central nervous system (CNS) [34]. Skin involvement, arthritis and renal disorder are very common manifestations in patients with SLE. Among them, skin involvement generally manifests as alopecia, mucosal ulcers or malar rash. Renal disorders range from asymptomatic hematuria or proteinuria to overt nephritic and nephrotic syndromes, rapidly progressive glomerulonephritis, and chronic renal failure [35]. So far, the pathogenesis of SLE has not been illuminated clearly.

Functions of IFN-λ1, a type III IFN, include inhibition of viral infection and proliferation of tumor cells as well as regulation of immune system [10, 12‐18, 36]. To the best of our knowledge, the role of IFN-λ1 in the progression of SLE remains unknown. Therefore, we hypothesized that IFN-λ1 played a role in the pathogenesis of autoimmune diseases such as SLE. Real-time PCR and ELISA assays were used to detect IFN-λ1 mRNA expression in PBMC and serum IFN-λ1 protein levels, respectively. Our initial data showed that IFN-λ1 mRNA expression and serum protein levels in patients with SLE were higher compared with normal controls, suggesting a role for IFN-λ1 in the pathogenesis of SLE.

We were also interested as to whether IFN-λ1 was associated with disease activity. Under these circumstances, SLE patients were divided into active and inactive groups according to SLEDAI scores, and active groups were defined as a SLEDAI score ≥6 [33]. By analyzing the data of different groups, finding that IFN-λ1 mRNA and protein levels in patients with active disease were significantly higher compared with patients with inactive disease together with normal controls. Because we have known that IFN-λ1 can be secreted by PBMC as well as DC [2, 8], meantime, SLE is a kind of autoimmune disease characterized by massive abnormal immune cells response which leads to autoimmune disorders and dysregulation. Therefore, we speculated that elevated IFN-λ1 mRNA and protein levels in patients with SLE probably were related to abundant and inordinate immune cell response. Thus, our findings implied that IFN-λ1 probably involved in the disease activity of SLE.

Moreover, we made further efforts to analyze the correlation between serum IFN-λ1 protein levels and SLEDAI together with several laboratory values, such as anti-dsDNA, AnuA, Smith, rRNP, AHA antibody, ESR, CRP, C3, C4 and 24-hour urine protein. Our analysis results disclosed that significant positive correlations were found between serum IFN-λ1 levels and SLEDAI, anti-ds-DNA antibody and CRP, and there was also a negative relationship between IFN-λ1 levels and C3. CRP is an acute-phase protein known as a biomarker for inflammation, and has been traditionally used to detect and predict the outcome of infections, inflammatory, necrotic processes as well as monitor clinical disease activity together with efficacy of treatment [37, 38]. Reports have displayed that CRP levels above 60 mg/l in febrile SLE patients without serositis almost always indicate infection; whereas in SLE alone, CRP levels are only moderately raised even in patients with very active disease [39]. In our study, the highest CRP value was 26.9 mg/l, and most CRP values were between 0 to 10 mg/l; therefore, in this study, CRP was a biomarker not indicating infection but monitoring disease activity. C3, one of complement components, participates in elimination of immune complexes through combination with immunoglobulin to disturb interaction of crystallizable fragment in space, and its reduction indicates disease activity. Therefore, by these results, we obtained the conclusion that IFN-λ1 may influence disease activity in SLE for a second time.

In patients with SLE, various manifestations make patients feel pain both physically and mentally. The most common clinical manifestations were arthritis (67%), malar rash (66%), nephritis (55%), and central nervous system (CNS) disease (27%) [40]. In consequence, we also assessed the correlation between IFN-λ1 and disease manifestations in patients with SLE; however, we did not distinguish the relationships among serum IFN-λ1 protein levels and alopecia, mucosal ulcer, malar rash, fever, chest affection, neurological disorder, anemia, thrombocytopenia and leucopenia. We discriminated that serum IFN-λ1 protein expression was significantly higher in patients with renal involvement and arthritis in comparison with patients without the above-mentioned disease manifestation as well as normal controls. In this case, we had evidence to infer that IFN-λ1 may involve in development of renal involvement and arthritis in SLE. Although our research did not observe an association between IFN-λ1 protein expression and other clinical manifestations, maybe this result was related to the limitation of small samples.

Since we had come to a decision that IFN-λ1 may participate in the pathogenesis of SLE, and has association with disease activity as well as progression of arthritis and renal disorder, we were interested in the mechanism by which IFN-λ1 played an important part in the development of SLE.

Chemokines are a group of molecules which have the ability to direct leucocytes to inflammatory sites to take part in immune response, influence maturation of kinds of immune cells, such as DC, T cell and stimulate monocytes, NK cells as well as T cell migration together with accommodate adhesion molecule expansion [20‐22]. Chemokines IP-10, MIG and IL-8 are from the CXC family, and play an important role in the chronic inflammatory immune responses by recruiting leukocytes to inflammatory sites [25‐27].

Besides, recent studies have reported that serum inflammatory chemokines IP-10 and MIG levels were elevated in SLE patients compared with normal controls. In addition, they had a positive relation with SLEDAI. Furthermore, in SLE patients with renal involvement, IP-10 and IL-8 levels were positively correlated with disease activity [29]. Beyond that, IP-10, IL-8 and MIG levels in cerebrospinal fluid (CSF) were significantly increased in neuropsychiatric SLE (NPSLE) patients compared with those with non-NPSLE and nonautoimmune diseases [41]. Another research team had calculated chemokine scores using several chemokines containing IP-10, MIG, IL-8 and other chemokines, finding that chemokine scores were significantly elevated in SLE patients versus RA patients and healthy donors and were correlated positively with SLEDAI scores and negatively with C3 levels. Compared with patients without lupus nephritis and those with inactive lupus nephritis, chemokine scores were increased in patients with active lupus nephritis. Elevated chemokine scores were also associated with the presence of cumulative organ damage and the occurrence of anti-Sm or anti-RNP autoantibodies [42]. Thus, elevation of chemokines could promote immune cells to migrate to inflammation site and take part in progression of inflammation by their cytotoxic effect or secretory inflammatory mediator in autoimmune diseases including SLE.

What is more, Pekarek V [36] had reported that IFN-λ1 could elevate mRNA levels of MIG, IP-10 and 'IFN-gamma inducible T-cell alpha chemoattractant' (I-TAC/CXCL11) from normal human PBMC. As a result, for exploring the function mechanism of IFN-λ1 in SLE, we detected the ability of IFN-λ1 to induce PBMC to secret MIG, IP-10 and IL-8 in patients with SLE, recognizing that in patients with SLE, IFN-λ1 had the ability to induce secretion of IP-10, MIG and IL-8. Meanwhile, it had its effects at three different concentrations with MIG levels increasing dose-dependently. Moreover, several concentrations of IFN-λ1 had a synergistic role with LPS in the production of chemokines IP-10, MIG and IL-8. Consequently, we deduced that IFN-λ1 presumably took effect in SLE by stimulating overexpression of chemokines IP-10, IL-8 and MIG associated with pathogenesis of SLE. In this case, it is reasonable to infer that after producing by a mount of abnormal PBMC, IFN-λ1 then stimulated PBMC to secret chemokines which could participate in the development of SLE through several different mechanisms. Therefore, this condition becomes a sort of vicious circle which is harmful to the disease situation of patients with SLE.

We know that bacterial infections may serve as environmental triggers for the development or exacerbation of SLE in genetically predisposed individuals [43], and that bacterial LPS plays an important role in the pathogenesis of SLE, such as, it could aggravate disease development by activating proliferation of B cells, production of autoantibodies and proinflammatory cytokines [44, 45]. Moreover, Pawar RD et al. [46] reported that in nephritic MRL ^lpr/lpr mice, transient exposure to bacterial cell wall components LPS increased splenomegaly, production of DNA autoantibodies, serum IL-6, IL-12 as well as tumor necrosis factor (TNF) levels, and aggravated lupus nephritis (LN), which was a major complication of SLE and associated with high rates of morbidity [43]. In our study, we observed that there was a synergic effect of IFN-λ1 and LPS on the chemokines expression. Thus, we inferred that, except for the various effect of single LPS on the development of SLE and LN, together with the synergic effect of IFN-λ1 and LPS on the chemokines secretion, they could play a powerful effect on the inflammation process of SLE, and promote the disease aggravation in patients with SLE, especially with LN. Thus, we supposed that not only did IFN-λ1 participate in the renal involvement but also it played the pathogenic role by combining with the effect of LPS. Therefore, for patients with SLE accompanying LN, they should avoid bacterial infections in order to prevent disease progression.

At the same time, it is interesting to compare the role of different types of IFN in the pathogenesis of SLE. As is well known, Type I IFN (IFN-α, IFN-β) and Type II IFN (IFN-γ) are classic interferons, and play an important role in the pathogenesis of SLE. Some reports have displayed that compared to healthy and unrelated individuals, higher activity of IFN-α was found in the serum of both SLE patients and healthy relatives, and it was associated with autoantibodies to RNA-binding proteins and double-stranded DNA [47]. Levels of Type I IFN correlated with the presence of cutaneous manifestations, and positively with the SLEDAI score and anti-dsDNA levels and inversely with C3 levels [3]. Expression of IFN-γ was significantly increased in the PBMC of SLE patients compared with healthy controls [5], and its expression in urinary sediment was significantly higher in the active lupus nephritis than in inactive SLE and previous renal involvement. Among the SLE patients, there was a close correlation between expression of IFN-γ and the SLEDAI score [6]. Therefore, compared with the above mentioned research findings, we found that there was a lot of similarity with respect to the role of IFN-λ1 and Type I IFN as well as Type II IFN in the pathogenesis of SLE.

However, we must acknowledge some limitations of this study. The sample size of our study was small with 42 lupus patients and 25 controls, because a small sample size has a greater probability that the observation may happen to be especially good or bad, and statistical tests usually require a larger sample size to justify that the effect did not happen by chance alone. Moreover, owing to its cross-sectional design, it is difficult to establish the exact and definite causal relationships except association between IFN-λ1 and development of SLE from the collected data. Another disadvantage of such a study is that it can only identify a high proportion of prevalent cases of long duration. Patients who die soon or who recover quickly are less likely to be identified as diseased.

Meanwhile, because the mechanism of IFN-λ1 in the pathogenesis of SLE is very complicated, we hope there will be a lot of new findings about the role of IFN-λ1 in autoimmune diseases including SLE in the near future.

In conclusion, this preliminary study demonstrated that IFN-λ1 perhaps played a part in the pathogenesis of SLE and had a positive association with disease activity. Meanwhile, it was likely to refer to the development of renal disease as well as arthritis in SLE. Furthermore, we observed that IFN-λ1 probably played an important role in the pathogenesis of SLE by inducing production of chemokines IP-10, MIG and IL-8 which attended the progression of SLE. The present study of IFN-λ1 may offer new insight for future studies on pathogenesis of SLE and novel research targets for SLE therapy.

The present study suggests that IFN-λ1, as one kind of interferon, may participate in the pathogenesis of SLE by modulating the expression of chemokines IP-10, MIG and IL-8, and IFN-λ1 may be related to disease activity in SLE. The findings of such studies will provide new insight into the pathogenesis as well as therapy of SLE, and will shed new light on the dysregulation of the immune system in autoimmune diseases.