5-aminolevulinic acid combined with sodium ferrous citrate ameliorated lupus nephritis in a mouse chronic graft-versus-host disease model
Introduction
Systemic lupus erythematosus (SLE) is a systemic autoimmune disease characterized by the abnormal activation of immune cells and hypersecretion of autoantibodies by dysfunction autoreactive B cells and causes irreversible chronic damage and dysfunction of body organs, such as joints, skin, kidneys, heart, lung and blood vessels [1], [2]. Lupus nephritis is the most common complication to cause death in patients, currently afflicting up to 60% of SLE patients [3]. Immune complex-mediated glomerulonephritis is caused by the deposition of immune complexes and autoantibodies in the kidneys. It is the most common and serious clinical manifestation of SLE and can cause extremely high mortality in affected individuals [4]. Currently available treatments include immunosuppressants, biological agents and glucocorticoids. Among them, B cell depletion using rituximab has been used to treat SLE and many autoimmune diseases and chronic inflammatory diseases; however, because this therapy cannot remove long-lived plasma cells, its role is limited. Furthermore, none of these therapies are permanent or able to reverse immune imbalances [5], [6].
Among the mouse models of SLE disease, chronic graft-versus-host disease (cGvHD) is a recognized lupus model that represents a common mouse model of SLE-like disease, it can be induced by transferring CD4+ T cells into MHC-II mismatched recipients, an established model in which parental DBA/2 spleen cells are injected into a B6D2F1 recipient. The model is characterized by SLE-like disease accompanied by abnormal B cell expansion, autoantibody production and glomerulonephritis. Donor CD4+ T cells activate and expand host B cells with autoreactive potential, leading to human SLE-like disease conditions, such as a high titer of autoantibody production and lupus nephritis. Although the cellular and molecular events leading to the destruction of tolerance and the appearance of pathological autoantibodies are still rather vague, genetic characteristics clearly play a key role in susceptibility to SLE [7], [8].
5-aminolevulinic acid (5-ALA), commonly found in plants, bacteria, fungi and animals, is a precursor to biosynthetic tetrapyrroles, including chlorophyll, Vitamin B12, and heme. 5-ALA is an endogenous amino acid in animals and is the first compound produced by 5-ALA synthase in the heme biosynthetic pathway [9], [10], [11]. Several studies have shown that 5-ALA has antifibrotic, antitumor, anti-inflammatory and antioxidative effects. Our previous studies showed that the heme oxygenase (HO)-1 expression was up-regulated by 5-ALA and sodium ferrous citrate (SFC) [12], [13]. HO-1 is known to be a rate-determining enzyme in heme metabolism that produces bilirubin and carbon monoxide, which were shown to act as anti-inflammatory factors [14], [15], [16]. However, few studies have focused on the ability of 5-ALA/SFC to inhibit immune cell activation. Our group has used 5-ALA/SFC as an immunosuppressant in different mouse models of immune disease, including murine sclerodermatous as a cGvHD model and models of cardiac allografts [17], [18]. Furthermore, 5-ALA/SFC can function as an immunosuppressive therapy by inducing Tregs. While HO-1 stimulates the immune response by expanding the population of effector T cells, it has also been reported to be involved in T cell activation and can limit immune-based tissue damage through Treg suppression, which promotes effector response [17], [19]. Therefore, this property makes HO-1 an important regulator of peripheral self-tolerance by balancing the ratio of effector T cells to Tregs. Interestingly, the T cells of some SLE patients proved to be highly activated, although these patients also showed fewer Tregs than did healthy individuals [20], [21]. Therefore, the impairment of HO-1 production in some SLE patients may be the cause of Treg homeostasis, resulting in a decreased tolerance to self-antigens.
In the present study, we investigated the therapeutic effects of 5-ALA/SFC on lupus nephritis in a cGvHD model and conducted experiments, including assessing the in vivo effect of 5-ALA/SFC on serum autoantibodies, immune cells and the expression of inflammatory cytokine mRNA. Our findings demonstrated that Treg numbers were increased by 5-ALA/SFC treatment and largely protected mice against cGvHD. Furthermore, the therapeutic administration of 5-ALA/SFC at three weeks (in the early stage of the disease) led to the significant inhibition of the stimulation of donor CD8+ T cells and B cells and the increased expression of HO-1 by donor Tregs.
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Mice
Seven- to eight-week-old female C57BL/6NJcl x DBA/2NJcl (B6D2F1, H-2kb/d) and DBA/2N (DBA, H-2kd) mice were purchased from CLEA Japan, Inc. (Tokyo, Japan). Since female mice are more susceptible than males for inducing cGVHD [22], we used female mice in this study. Mice were maintained on a 12-h light‐dark cycle and given free access to food and water except during the period of caloric restriction for the pair‐feeding group. All animal manipulations were performed according to the
Effect of 5-ALA/SFC on lupus nephritis in a mouse cGVHD model
To determine the therapeutic effect of 5-ALA/SFC on lupus nephritis mice, we used a mouse cGvHD model, which was established by transferring 5 × 107 spleen cells from donor DBA (H-2kd) into recipient B6D2F1 (H-2kb/d) mice; recipient mice were then treated with 5-ALA/SFC orally administered at 100 mg/kg and 157 mg/kg daily. As shown as in Fig. 1A, B, recipient mice develop a typical lupus syndrome characterized by significantly increased levels of serum BUN, Cre and dsDNA autoantibody at
Discussion
Some SLE mouse models, such as New Zealand strains and MRL/lpr mice, mainly due to loss of immune tolerance due to genetic variation [20]. The mouse cGvHD model used in the present study is easier to regulate and has a shorter cycle than the genetic mouse model. In addition, female donors will make female hosts more likely to develop SLE than males [24], [25].
Although the beneficial effects of ciclosporin A and other immunosuppressive agents on autoimmunity have been established for some time,
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgements
The authors thank Yuko Sato for her invaluable technical assistance. This study was supported by research grants from the Grants of Ministry of Education, Culture, Sports, Science and Technology of Japan (Grants-in-Aid 16K11064, 24/17H04277, 18K08558); and grants from the National Center for Child Health and Development (26-22).
Authors' contributions
LC, WZG, XH, HI, KT, MN, TT, ZP and XKL conceived and designed the project; LC, WZG, HX acquired the data; LC, WZG, XH, ZP and XKL analyzed and interpreted the data; LC and XKL wrote the paper. All authors read and approved the final manuscript.
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Current address: Reproductive & Women-Children Hospital, School of Medical and Life Sciences, Chengdu University of Traditional Chinese Medicine, Chengdu, China.