Background
Systemic lupus erythematosus (SLE) is a chronic inflammatory autoimmune disease [
1,
2], and lupus nephritis (LN) is one of the most severe SLE-associated organ complications [
3,
4]. The etiology of SLE has been still unclear, however, abnormalities of genetic variants, that of molecular pathways, and dysregulation of several immune cells have been previously reported as disease pathogenesis in SLE. Dysregulation of B cell receptor (BCR) signaling that can lead to autoimmunity including autoantigen presentation to activate T cells and pro-inflammatory signaling by cytokines secretion and complement activation have been implicated in pathogenesis of SLE and LN [
5,
6]. Especially, several pathogenic autoantibodies secreted by abnormalities of B cells play crucial roles in pathogenesis of SLE. Moreover, autoantibodies and intrarenal complement activation contribute to the renal immune-pathogenesis of LN [
7‐
9]. Given the crucial role of B cells in SLE pathogenesis, they have emerged as promising new targets for SLE and LN treatment in the last decade. Clinical trials of several B cell-targeted biological treatments, such as the anti-CD20 antibody rituximab [
10‐
12], B cell-activating factor inhibitor belimumab [
13], and anti-CD22 antibody epratuzumab [
14,
15], have been conducted, but only belimumab has demonstrated a clinical benefit, leading to US Food and Drug Administration approval [
16,
17].
Bruton’s tyrosine kinase (BTK) is a non-receptor tyrosine kinase belonging to the tec protein tyrosine kinase (TEC) kinase family that is encoded by the
TEC gene. BTK plays critical roles in activation mediated by the B cell receptor (BCR), Fc receptor (FcR), Toll-like receptor (TLR), and chemokine receptor [
18‐
20]. In humans, BTK deficiency causes inherited X-linked agammaglobulinemia [
21]. In mice, it leads to X-linked immunodeficiency (
xid) [
21] and BTK-knockout mice carrying the 56R anti-DNA Ig transgene do not produce anti-DNA antibodies [
22]. Moreover, macrophages in BTK-deficient
xid mice have impaired proinflammatory cytokine generation because BTK plays a major role in immune complex-mediated activation via FcγR in macrophages [
21]. BTK is closely associated with SLE and LN development via both BCR and Fc receptor signaling, and therapeutic potential of BTK inhibitors have been demonstrated in several SLE animal models. For example, it have been reported that the therapeutic effects for glomerular nephritis of RN486 and PF-06250112, selective BTK inhibitors, by inhibition of effector cells and target autoantibodies in NZB/W F1 mice with Fc receptor dependent and antibody mediated LN [
23,
24]. Ibrutinib also ameliorated humoral and cellular autoimmunity by partial crippling of cell signaling in both B cells and antigen presenting cells in lupus–prone B6.Sle1 and B6.Sle1.Sle3 mice [
25]. Thus, targeting the BTK signaling pathway may provide an effective therapeutic strategy [
26].
HM71224, orally active and irreversible small-molecule BTK inhibitor, may covalently bind to the active site (cysteine 481 residue) of BTK. HM71224 inhibited BTK, BMX, TEC and TXK which carry a conserved cysteine in the binding pocket in more than 85-kinase assays. HM71224 showed a highly selective inhibition for BTK with IC
50 of 1.95 nM. The selectivity toward other BMX, TEC and TXK were 0.3, 2.3 and 2.4 fold, respectively. Moreover, HM71224 completely occupied to BTK and revealed potent inhibition of BCR, FcR and TLR mediated signaling [
27].
In this study, to determine whether HM71224 could attenuate SLE and LN by suppressing BTK activation, the MRL/
lpr and New Zealand Black/White F1 (NZB/W F1) mouse models, which have physiological relevance for human lupus, were used. MRL/
lpr mice are homozygous for the lympho-proliferation spontaneous mutation (Fas
lpr
) and have impaired central tolerance [
28,
29], and NZB/W F1 mice co-express several
sle loci that are associated with increased risk of SLE development [
30].
Methods
Immunoblotting
Ramos cells (American Type Culture Collection, Manassas, VA, USA), the human Burkitt’s lymphoma cell line, were cultured in suspension using Roswell Park Memorial Institute (RPMI) 1640 medium (Gibco, Rockville, MD, USA) with 10% fetal bovine serum (Gibco) at 37 °C in 5% CO2 in air. Cells were starved of serum for 1 h and then treated with HM71224 for 1 h. Cells were washed with phosphate-buffered saline (Welgene, Korea) and then were stimulated with 1 μg/ml of goat F(ab’)2 anti-human IgM (Southern Biotech, Birmingham, AL, USA) for 10 min on ice. Cells were then lysed in radioimmunoprecipitation assay (RIPA) buffer (Sigma-Aldrich, St. Louis, MO, USA) and the proteins were separated by SDS-PAGE (Bio-Rad Laboratories, Hercules, CA, USA) and transferred to polyvinylidene fluoride membranes (EMD Millipore, Billerica, MA, USA). Proteins were detected with anti-BTK Y223 (Cell Signaling Technology, Danvers, MA, USA), anti-BTK (53/BTK, Cell Signaling Technology), anti-PLCγ2 Y1217 (Cell Signaling Technology), anti-PLCγ2 (Cell Signaling Technology), and anti-GAPDH (Santa Cruz Biotechnology, Santa Cruz, CA, USA) antibodies. Protein signals were visualized by chemiluminescence using ECL western blotting detection reagent (EMD Millipore), and scanned blots were quantified using a Multi-Image analyzer (Fujifilm, Japan). The percentage of phosphorylation of each lane was determined using Multi-Gauge software (Fujifilm). The IC50 value was calculated using control IgM+ set at 100% and control IgM- set at 0%.
FcγR-stimulated cytokines
THP-1 cells (American Type Culture Collection) were differentiated to macrophages by stimulation with interferon-γ (IFN-γ) at 10 ng/ml in RPMI 1640 medium (Gibco) with 10% fetal bovine serum (Gibco) for 6 days at 37 °C in 5% CO2 in air. Monocyte-derived macrophages were pre-treated with HM71224 for 30 min. Cells were stimulated with a high concentration of plate-bound human IgG. The supernatant was collected after 24 h, and TNF-α and IL-6 were measured using commercially available ELISA kits (R&D Systems, Minneapolis, MN, USA).
Experimental protocol for MRL/lpr mice and NZB/W F1 mouse models
Female MRL/MpJ-Fas
lpr/J mice (20 ± 5 g, 8 weeks old) and female NZB/W F1 mice (30 ± 5 g, 18 weeks old) were obtained from SLC Inc. (Japan). Animals were housed and handled in a temperature-controlled environment with a 12-h light/12-h dark cycle. They had free access to standard pelleted food (Picolab Rodent diet 5053, St. Louis, MO, USA) and water
ad libitum. In MRL/
lpr mice, 12 animals per group identified by their urine protein score [
31] were treated with HM71224 orally once per day from 8 weeks through 28 weeks of age, and in NZB/W F1 mice, 12 animals per group were administered HM71224 orally once daily from 18 weeks through 40 weeks of age. All animal experimental protocols and procedures were approved by the Institutional Animal Care and Use Committee of the Hanmi Research Center and performed according to the approved guideline.
Skin lesion scores
Macroscopic lupus-erythematosus-like skin lesions were evaluated only in MRL/l
pr mice with the spontaneous development of skin lesions similar to those seen in human SLE [
32]. Skin lesions were scored every week and were expressed using a scoring system from 0 to 3 (0, none; 1, mild; 2, moderate (<2 cm); 3, severe (≥ 2 cm)) for changes in the nose, ears, and intrascapular region.
Urine protein
Urine protein was individually evaluated weekly or biweekly using a urine test strip (URiSCAN strips, YD Diagnostics, Korea). Scores for protein concentration were graded from 0 to 4: 0, none; 1, 30–99 mg/dl; 2, 100–299 mg/dl; 3, 300–1999 mg/dl; 4, ≥ 2000 mg/dl or death.
Blood urea nitrogen, creatinine, and autoantibody measurements
Blood samples were obtained from mice via the caudal vena cava under anesthesia with ether. Blood was centrifuged at 13,400 g for 2 min at 4 °C to collect serum. Blood urea nitrogen (BUN) and creatinine (CRE) were measured using a Hitachi 7020 automatic chemical analyzer (Hitachi, Japan). Serum mouse anti-dsDNA antibody levels were determined using a commercially available ELISA kit (Alpha Diagnostic, San Antonio, TX, USA) according to the manufacturer’s instructions.
Kidney histopathological analysis
The kidneys were surgically removed under anesthesia with ether. They were fixed in 10% neutral buffered formalin and embedded in paraffin. Sections of 4-μm thickness were stained with hematoxylin and eosin and Periodic acid Schiff. The histopathological score was evaluated microscopically in a blinded manner. The membranous glomerulonephritis score (GN score) was evaluated using scoring from 0 to 4: 0, normal; 1, mild, focal or early proliferation; 2, moderate or definite proliferation and increased matrix; 3, diffuse and focal or diffuse proliferation; 4, severe diffuse proliferation with crescent/sclerosis. The renal interstitial nephritis score (IN score) was graded from 0 to 4 for inflammation and necrosis: 0, normal; 1, occasional, focal or small pockets of mononuclear cells (MNCs, 10–14 cells); 2, focal infiltration of MNCs (15–30 cells); 3, multifocal and extensive infiltration of MNCs; 4, severe infiltration of MNCs with extensive necrosis. Vasculitis was scored from 0 to 4: 0, normal; 1, occasional perivascular infiltration of MNCs; 2, several foci of perivascular infiltration of MNCs without necrosis; 3, multifocal perivascular infiltration of MNCs with/without necrosis; 4, multifocal or diffuse perivascular infiltration of MNCs; extensive with necrosis.
Splenomegaly and lymphadenopathy
Spleen and cervical lymph nodes were removed and their weights were measured before fixation.
Flow cytometry
A single cell suspension from the spleen was prepared by homogenizing the spleen through a 40-μm mesh nylon cell strainer (BD Falcon 352340, BD Biosciences, San Jose, CA, USA). Red blood cells were lysed using ACK lysing buffer (Gibco). Splenocytes were stained with fluorescein isothiocyanate (FITC)-conjugated rat anti-mouse CD45R/B220 (BD Biosciences), phycoerythrin (PE)-conjugated hamster anti-mouse CD69 (BD Biosciences), PE-conjugated rat anti-mouse GL7 (BD Biosciences), and PE-conjugated rat anti-mouse CD138 (BD Biosciences). At least 10,000 stained cells were counted and were analyzed with FACSCalibur™ flow cytometry (BD Biosciences). All splenocytes were primarily gated on live lymphocytes based on forward scatter (FCS) and side scatter (SSC). Germinal center B cells were identified as B220+ and GL7+. Activated B cells were identified as B220+ and CD69+, and plasma B cells were identified as B220+ and CD138+.
Statistical analysis
Data are expressed as the mean ± SEM. Analysis of the significance of differences was performed using one-way analysis of variance (ANOVA) (for parametric data) or the Kruskal–Wallis test (for non-parametric data) using GraphPad Prism (version 5.0, La Jolla, CA, USA). In Kaplan-Meier survival curve, the curve comparison was resulted of the log-rank test using GraphPad Prism.
Discussion
B cell hyperactivation and immune complex-mediated FcR activation are known to play a major role in the pathogenesis of immune-mediated LN [
5‐
9]. A previous report demonstrated the benefits of BTK inhibition in mouse models of TLR7/IFN-driven lupus by affecting both BCR and FcR signaling [
23]. We also have previously demonstrated that BTK inhibition by HM71224 effectively blocks not only the phosphorylation of both BTK and PLCγ2 through downstream BCR signaling in human B cells, but also FcR-stimulated cytokine production in human monocytes [
27].
Our present study demonstrates that HM71224 effectively reduced the numbers of several splenic B cell types. Germinal centers are central to development of long-lived plasma B cells, their clonal expansion, and somatic hypermutation affinity maturation. In SLE a large proportion of these autoreactive plasma cells, which are generated in the spleen, preferentially migrate to the inflamed kidneys. Therefore, the spontaneous formation of these germinal centers, which are an important source of autoreactive plasma cells, is considered a hallmark of SLE, and disease progression of LN is associated with an increase in the number and size of germinal centers [
33,
34]. In this study, HM71224 dose-dependently and considerably decreased the number of splenic B220
+GL7
+ germinal center B cells, and this inhibition of B cell differentiation for germinal center formation may exert a therapeutic effect on LN. Inflamed tissue and secondary lymphoid organs can contribute to the number of autoreactive plasma cells in SLE [
35]. In this study, HM71224 dose-dependently and significantly decreased the number of splenic B220
+CD138
+ plasma cells, which are considered the major producers of autoantibodies. Moreover, HM71224 strikingly inhibited splenic B220
+CD69
+ B cell numbers, which are early markers of B cell activation and antigen presentation. This result is consistent with previously reported data showing that B cells with defective BCR signaling fail to mature and are non-responsive to BCR cross-linking in terms of proliferation and upregulation of the activation marker CD69 [
36,
37]. These results suggest that HM71224 may effectively inhibit autoantibody production and cytokine enhancement by B cells, which are caused by presenting autoantigens to CD4
+ T cells as antigen-presenting cells and stimulating dendritic cells.
These numerous inhibitory effects of splenic B cells are consistent with the prevention of splenomegaly and enlargement of cervical lymph nodes observed following treatment with HM71224. Approximately 50% of patients with SLE have enlarged lymph nodes and these are usually detected in the cervical, axillary, and inguinal areas. In particular, lymphadenopathy is more frequently noted at the onset of disease or during disease flares [
38]. Splenomegaly also occurs in 10–45% of patients with SLE owing to disordered immunoregulation and is particularly observed during active disease [
39]. Thus, inhibition of both lymph node enlargement and splenomegaly are considered to be positive signals of the suppression of SLE-like disease progression by HM71224 in an animal model of lupus.
The first major therapeutic finding was that treatment with HM71224 resulted in no skin lesions in MRL/
lpr mice. Skin rash is the second most common clinical manifestation in patients with SLE and in MRL/
lpr mice, which represent a unique mice strain, with development of a skin rash [
40]. The pathogenesis of skin lesions, hair loss, and scab formation in MRL/
lpr mice is characterized by immunoglobulin and/or complement depositions and accumulation of various other components of the immune system including macrophage/monocytes and dendritic cells [
41,
42]. The B cell deficient and spleen tyrosine kinase (SYK) inhibitor-treated MRL/
lpr mice were found not to develop spontaneous dermatitis [
8,
43] and rituximab has been shown to have a therapeutic effect against cutaneous lesions in patients with SLE [
44]. Here, we demonstrated that the typical skin lesions around the nose or eyes were observed in vehicle-treated mice but not in HM71224-treated mice, and these effects in skin lesions may be associated with B cell modulation by HM71224.
The second major therapeutic finding was that HM71224 had therapeutic effects related to the amelioration of kidney damage caused by lupus-like renal inflammation. Intrarenal B cells can contribute to renal damage and inflammation by enhancing the immunological response as antigen-presenting cells, inducing cytokine-promoting T cell proliferation and lymphatic angiogenesis, and enhancing the local immune response to persisting autoantigens in the tubulointerstitium [
45,
46]. In both MRL/lpr and NZB/W F1 mice, activation of TLR9 caused accelerated LN [
47]. On the other hand, LN develops via unique mechanisms in each strain like the highly heterogenous nature of SLE. NZB/W F1 mice promote the renal damages B cell dependent manner including secretion of autoantibody, whereas MRL/lpr mice develop the renal damages via antigen presentation and cytokine production by B cells, not secretion of autoantibodies [
5].
As discussed previously, HM71224 markedly reduced splenic B cell numbers, including those of germinal center B cells, plasma B cells, and activated B cells. We also reported the inhibitory effects of HM71224 on BCR and FcR signaling [
27]. These mechanisms of action of HM71224 might lead to significant amelioration of renal injury and lymphocyte infiltration in both animal models, although differences in strains in the pathogenesis of LN can lead to different degrees of therapeutic drug efficacy. Therefore, we suggest that the therapeutic effects of HM71224 in glomerulonephritis, interstitial nephritis, and vessel inflammation in murine LN were mediated by B cell inhibition.
Finally, one of the major therapeutic findings was that there was no mortality during the period of HM71224 administration in both mice models (Fig.
8). Prevention of renal damage by HM71224 might have led to the improvement in survival rate, because LN leads to proteinuria and renal failure and is associated with significant mortality [
48]. Although cyclophosphamide treatment has markedly reduced mortality in LN from more than 70% in the 1960s to less than 10% in recent years, mortality remains relatively high [
49]. Therefore, despite continuous improvements in SLE therapeutics, mortality remains a serious problem in LN therapy [
3,
4]. Thus, the most notable result of this study is that HM71224 can markedly increase the survival rate of animals with SLE by preventing renal damage and inflammation.