Suppression of lupus nephritis and skin lesions in MRL/lpr mice by administration of the topoisomerase I inhibitor irinotecan
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Since the precise mechanism for the pathogenesis of systemic lupus erythematosus (SLE) is unknown, no targeted therapies in addition to immunosuppression are available so far. We recently demonstrated that administration of the topoisomerase I (topo I) inhibitor irinotecan at extremely low concentrations reversed established lupus nephritis in NZB/NZW mice. While profound immunosuppression was absent, we proposed changes in DNA relaxation and anti-double-stranded (ds)DNA antibody binding as the underlying mechanism. To exclude that these effects were restricted to NZB/NZW mice, irinotecan was used in a genetically different strain of lupus-prone mice.
MRL/lpr mice were treated with high- and low-dose irinotecan beginning at 8 weeks of age. Treatment was repeated every fourth week. In vitro, DNA was relaxed by recombinant topo I, and altered anti-dsDNA antibody binding was measured by enzyme-linked immunosorbent assay.
Administration of both high- and low-dose irinotecan prevented proteinuria and prolonged survival in MRL/lpr mice. Moreover, both concentrations of irinotecan significantly improved histopathology of the skin at 18 weeks of age. While only high-dose irinotecan diminished the numbers of plasmablasts and double-negative T cells, no changes in IgG-secreting cells or anti-dsDNA IgG were observed. In vitro, relaxation of DNA by topo I increased the binding of anti-dsDNA IgG but not the binding of anti-dsDNA IgM derived from the plasma of MRL/lpr mice.
The beneficial effects of topo I inhibition in a second, genetically different strain of lupus-prone mice strongly implicate irinotecan as a new therapeutic option for human SLE.
KeywordsSystemic lupus erythematosus (SLE) Lupus nephritis Lupus-like skin lesions Alternative treatment for SLE Inhibitors of topoisomerase I DNA relaxation Anti-dsDNA binding
Analysis of variance
Bovine serum albumin
Enzyme-linked immunosorbent assay
Enzyme-linked immunosorbent spot
End-stage renal disease
Fetal calf serum
New Zealand Black/New Zealand White
Systemic lupus erythematosus
- topo I
Systemic lupus erythematosus (SLE) is a chronic autoimmune disease mainly affecting women of childbearing age. It is estimated that, in the USA, up to 275,000 adult women suffer from SLE . The disease involves different organs, but immune complex glomerulonephritis most strikingly influences the course of SLE. Ten to thirty percent of patients with lupus nephritis progress to end-stage renal disease (ESRD) resulting in hemodialysis or kidney transplantation . Due to the application of immunosuppressive drugs, the survival of patients with lupus-associated glomerulonephritis increased from a 5-year survival of 44 % in the 1950s to a 10-year survival of 88 % recently . Despite these advances in the treatment of SLE, the life expectancy of patients with lupus and renal damage was recently demonstrated to be 23.7 years shorter compared to the general population . Moreover, the incidence of ESRD associated with lupus nephritis has not decreased over the last years , indicating that current medication is insufficient to treat lupus nephritis.
Unselective immunosuppressive drugs remain the central strategy to control lupus nephritis. Medication consists of an induction therapy with cyclophosphamide and prednisolone or mycophenolate mofetil, followed by a maintenance therapy with azathioprine or mycophenolate mofetil [6, 7]. Major side effects of this medication are infections, and they bear the risk of malignancies whereupon cyclophosphamide also causes amenorrhea [8, 9, 10]. Furthermore, none of the newly developed biological agents such as rituximab, ocrelizumab, atacicept, or abatacept demonstrated beneficial effects in patients with active lupus nephritis [11, 12, 13, 14, 15]. Two other biologicals reached statistical significance for the improvement of moderate to severe forms of SLE. However, the benefit of belimumab and tabalumab were modest [16, 17], the “number needed to treat” was high, and other phase III studies hardly reproduced the first results [18, 19].
The precise mechanism for the pathogenesis of SLE is unknown. Therefore, no targeted therapies beside immunosuppression exist so far. However, a potentially targeted therapy for SLE was suggested by a new finding from our group which was generated by serendipity. We demonstrated that the topoisomerase I (topo I) inhibitor irinotecan efficiently suppressed murine lupus nephritis in New Zealand Black/New Zealand White (NZB/NZW) mice [20, 21, 22]. It was the first time that topo I was linked to the treatment of lupus nephritis.
Topo I is ubiquitously expressed and highly conserved [23, 24]. Its major function is the relaxation of supercoiled DNA in order to release torsional stress from DNA occurring during replication and transcription. To mediate DNA relaxation, topo I binds to DNA and cleaves one DNA strand, subsequently allowing the rotation of the cleaved strand around the other in a controlled reaction . Afterwards, the nicked strand is re-ligated by topo I restoring intact double-stranded (ds)DNA in a relaxed state.
Inhibitors of topo I stabilize a normally very transient catalytic intermediate in which topo I is bound to one strand of the DNA, known as the topo I cleavable complex. When the cleavable complex is stalled by topo I inhibitors, re-ligation of the DNA is impossible . The consequence of prevented re-ligation may vary; in proliferating cells, the stalled topo I cleavable complex can collide with replication forks leading to unrepairable DNA double strand breaks and apoptosis [27, 28]. For this reason, topo I inhibitors, like camptothecin and its synthetic derivative irinotecan, are widely used as anticancer drugs for several types of tumors . In non-dividing cells, the treatment with topo I inhibitors results in the production of single-stranded (ss)DNA breaks reducing the replication capacity of a cell, although this is not lethal . In addition to the induction of ssDNA and dsDNA breaks, topo I inhibitors were shown at least in vitro to inhibit DNA relaxation [30, 31].
In our first experiments, we applied concentrations of irinotecan that were similar to those used for chemotherapy in humans . Although irinotecan reversed established lupus nephritis, there have been some concerns about using yet another chemotherapeutic for the treatment of SLE in predominantly young women. However, subsequent experiments by our group demonstrated that much lower dosages were still efficient to suppress SLE. We ended up with a dose more than 50 times lower than the dose used for chemotherapy in humans, which still enabled the successful treatment of established lupus nephritis in NZB/NZW mice . These extremely low concentrations guided us to the hypothesis that inhibition of topo I might be a targeted therapy for SLE. While a profound immunosuppression was ruled out [20, 21, 22], we hypothesized in the beginning that induction of ssDNA breaks as a consequence of topo I inhibition is the underlying mechanism . However, later we found that topo I alone increased binding of anti-dsDNA antibodies [21, 22] supposing that DNA relaxation is critically involved in the pathogenesis of SLE.
All previous experiments using irinotecan for the treatment of SLE were performed in NZB/NZW mice. To provide more evidence for first clinical trials treating human SLE we had to rule out that the demonstrated effects of irinotecan are restricted to NZB/NZW mice. We therefore introduced the MRL/lpr mouse model which is characterized by a fast and severe disease progression involving fatal glomerulonephritis, vasculitis, skin lesions, and massive lymphadenopathy [33, 34]. In these mice, we tested whether irinotecan has similar beneficial effects on lupus-like disease as shown before in NZB/NZW mice.
Female MRL/lpr and MRL/MpJ mice, aged 6 weeks, were purchased from The Jackson Laboratory and kept in isolated ventilated cages. Immediately after arrival, mice were randomly assigned to the respective groups (five animals per cage).
Animal study: treatment of MRL/lpr with irinotecan
At 8 weeks of age, MRL/lpr mice were injected intraperitoneally with saline, or 1 or 25 mg/kg irinotecan (Campto®; Pfizer). MRL/MpJ mice treated with saline were used as controls. The volume of each injection was 10 ml/kg. Mice were treated three times per week. The treatment cycle was repeated after 4 weeks. Beginning at an age of 7 weeks, mice were monitored for proteinuria and body weight once a week. Proteinuria was measured with Albustix (Siemens Healthcare Diagnostics) and analyzed semiquantitively as grade 0 (negative), grade 1+ (≥30 mg/dl), grade 2+ (≥100 mg/dl), grade 3+ (≥300 mg/dl), and grade 4+ (≥2000 mg/dl) according to the manufacturer’s recommendations. The onset of proteinuria was defined as two instances of grade 4+ proteinuria occurring 1 week apart. Moreover, skin in the dorsal neck region, from the snout, and from the ears was scored individually in a semiquantitative manner using a score system from 0, for no lesion, to 2, for severe manifestation. Mice were killed when disease became severe (proteinuria grade 4+ and a body weight loss of ≥25 % from the onset of disease) and/or the total skin score was ≥4. The experiment was terminated when mice reached 37 weeks of age.
Histopathology of kidney and skin sections
Kidney and skin obtained from the dorsal regions were fixed overnight in 4 % paraformaldehyde and embedded in paraffin. Standard protocols were used for hematoxylin and eosin, periodic acid-Schiff, and methenamine-silver staining.
For cryosections, tissue was immediately placed in OCT, snap frozen in liquid nitrogen and stored at –80 °C. Sections (6-μm thick) were fixed in acetone for 10 min before incubation with Alexa Fluor 488-conjugated goat anti-mouse IgG (H + L chain specific; Invitrogen). The kidney score of glomerulonephritis was assessed by an independent pathologist who was blinded to the groups using the International Society of Nephrology/Renal Pathology Society 2004 classification . Skin was graded semiquantitatively according to Mizui et al. ; briefly, grade of acanthosis (none (0) to markedly thickened dermis (2)), hyperkeratosis (none (0) to strongly enhanced keratin (3)), fibrosis (normal (0) to markedly thickened dermal collagen (3)), inflammation (sparse (0) to substantial lymphocytic infiltrates (3)), and ulcer (absent (0) or present (1)).
Isolation of splenocytes and lung cells
Spleens were harvested from mice and immediately transferred into ice-cold phosphate-buffered saline (PBS), and smashed on a sterile grid with a pestle. Cells were incubated in red blood cell lysis buffer for 2 min on ice, debris was allowed to settle out by centrifugation at 65 g for 2 min at 4 °C, and cells were re-suspended in RPMI 1640 supplemented with 10 % fetal calf serum (FCS). Cell viability was checked by trypan blue exclusion.
The left lobe of the lung was minced to small pieces and digested in RPMI 1640 without supplement containing 100 μg/ml Liberase (Roche) for 90 min at 37 °C and 5 % CO2. Digestion was stopped by adding fetal bovine serum (Gibco) to a final concentration of 10 %. Cells were passed through a 40-μm cell strainer, incubated in red blood cell lysis buffer for 2 min on ice and resuspended in RPMI 1640 media for cell counting and further analysis.
Unspecific binding was blocked by incubating cells with Fc receptor-blocking monoclonal antibody (clone 2.4G2; BD Biosciences) for 10 min. Cells were then stained with the following antibodies specifically binding: CD3-PerCP/Cy5.5 (145-2C11; BioLegend), CD4-BV785 (RM4-5; BioLegend), CD8-BV421 (53-6.7; BioLegend), B220/CD45R-APC-Cy7 (RA3-6B2; BioLegend), CD138-BV605 (281-2; BioLegend), CD69-PE (H1.2 F3; BioLegend), and PD-1-PE-Cy7 (29 F.1A12; BioLegend). Dead cells were excluded by ZombieGreen™ (BioLegend) staining, and doublets by scatter analysis. Cells were measured on a LSRII flow cytometer (BD) and analyzed by FlowJo software. In most samples, a minimum of 1 × 105 events were measured.
Enzyme-linked immunosorbent spot (ELISpot) assay
Serial dilutions of splenocytes in RPMI 1640 supplemented with 10 % FCS were added to 96-well Multiscreen HTS Immobilon-P flat-bottomed plates (Millipore) pre-coated with goat anti-mouse IgG (Fc specific; Sigma-Aldrich) or goat anti-mouse IgM antibodies (BioLegend). After 4 h at 37 °C, plates were washed and incubated with alkaline phosphatase-conjugated anti-mouse IgG or alkaline phosphate-conjugated anti-mouse IgM (H + L chain specific, SouthernBiotech) for 1 h. Spots were developed with BCIP/nitroblue tetrazolium plus substrate (Sigma-Aldrich) and counted with an ELISpot reader (Autoimmun Diagnostika).
Enzyme-linked immunosorbent assay (ELISA)
Ninety-six-well Nunc MaxiSorp plates were coated with 5 μg/ml goat anti-mouse IgG (Sigma) or anti-mouse IgM (SouthernBiotech) overnight at 4 °C. Plates were blocked with 1 % bovine serum albumin (BSA) in PBS for 1 h at 37 °C followed by incubation with diluted plasma samples for 1 h at 37 °C. Subsequently, plates were incubated with alkaline phosphatase-conjugated goat anti-mouse IgG or anti-mouse IgM (SouthernBiotech) for 1 h at 37 °C and developed with p-nitrophenyl phosphate (Sigma-Aldrich). Optical density was measured at 405 nm with a reference filter at 490 nm. Sample concentrations were calculated using a standard curve of purified mouse IgG (Sigma) and IgM (SouthernBiotech).
To determine anti-dsDNA antibodies, calf thymus DNA (Invitrogen) was passed through a Millex-HA 0.45-mm filter (Millipore) to remove any ssDNA fragments. Maxisorp plates were half-coated with 100 mg/ml calf thymus DNA in PBS overnight at 4 °C. Plates were blocked with PBS containing 1 % BSA for 1 h at 37 °C. Diluted plasma samples were incubated at 37 °C for 1 h. Bound anti-dsDNA autoantibodies were detected as described above for total IgG or IgM ELISA, respectively.
Calf thymus DNA (50 mg/ml) was incubated with 4 μg/ml recombinant human topo I (Creative Biomart) in 50 mM Tris HCl (pH 7.5), 50 mM KCl, 2 mM dithiothreitol (DTT), and 1 mM EDTA for 30 min at 37 °C. Then, 30 ml of sample per well was used for coating a 384-well Nunc Maxi-Sorp plate overnight at 4 °C. Plates were blocked with PBS containing 1 % casein (Pierce) for 1 h at 37 °C. Next, depending on the experiment, diluted plasma samples from MRL/lpr mice were added to the plates for 1 h at 37 °C. Bound antibodies were detected as described above for anti-dsDNA IgG and IgM ELISA.
Data were analyzed using either one-way or two-way analysis of variance (ANOVA) followed by Bonferroni’s post-hoc test, respectively. Some data were assessed by paired t test. Survival data were analyzed using the Mantel-Cox log rank test. For all tests, the software GraphPad Prism version 6.0 was used. P values <0.05 were considered as significant.
Irinotecan attenuated symptoms of lupus-like disease and prolonged survival in MRL/lpr mice
MRL/lpr mice possess a rapid and severe form of lupus-like disease. For clinical monitoring, the involvement of the kidneys with proteinuria and the involvement of skin with alopecia, pruritus, and necrotic lesions are important. Both organ contributions influence the survival of these mice, in contrast to the NZB/NZW model preferably showing renal manifestations.
Excluding animals that were sacrificed due to the skin score, both high- and low-dose irinotecan demonstrated a significantly improved survival at 20 weeks of age (Fig. 1c). When including all animals, only MRL/lpr mice treated with high-dose irinotecan demonstrated an increased survival of 80 % compared to 0 % of saline-treated mice and 10 % of animals treated with low-dose irinotecan at 37 weeks of age (Fig. 1d).
Immunomodulatory effects of irinotecan treatment in MRL/lpr mice
B-cell numbers identified as CD3–B220+CD138– cells remained unchanged in both irinotecan-treated groups (Fig. 3c). By contrast, the expression of CD69 on B cells and the numbers of plasmablasts (CD3–B220+CD138int) were significantly decreased in MRL/lpr mice treated with high-dose irinotecan. For plasma cells (CD3–B220–CD138+), no significant effect of either high-dose or low-dose irinotecan compared to saline was observed (Fig. 3c).
High-dose irinotecan decreased the number of DN T cells infiltrating the lung
Increased binding of anti-dsDNA antibodies to DNA modified by topo I was restricted to the IgG isotype
Five years ago, our group reported for the first time that the topo I inhibitor irinotecan is able to suppress lupus nephritis in NZB/NZW mice , a finding that was based on serendipity obtained from cancer research experiments. In those experiments, immunocompetent mice were treated with a human protein either alone or in combination with irinotecan. During the second cycle of treatment, all mice from the group treated with the protein alone died within 3 days whereas all mice treated with the protein in combination with irinotecan survived. In the review of these unexpected results we speculated about the occurrence of some form of fatal hypersensitivity reaction and a protective effect of irinotecan against this. Based on this hypothesis we introduced a mouse model for anaphylaxis in terms of fast hypersensitivity reaction. However, irinotecan failed to delay the onset of anaphylactic shock (unpublished results). We next checked whether protection from delayed hypersensitivity reactions was involved and established two mouse models of spontaneous autoimmune disease: diabetes in NOD mice and SLE in NZB/NZW. While irinotecan had no effect on the development of autoimmune diabetes (unpublished data), application of the topo I inhibitor almost completely prevented the onset of lupus nephritis. Encouraged by this finding we also examined whether irinotecan ameliorates the course of two other autoimmune diseases, multiple sclerosis and systemic sclerosis. Both attempts failed (unpublished results) providing evidence that inhibition of topoisomerase I is a specific treatment for SLE. This suggestion was supported by further experiments showing that extremely low concentrations of irinotecan still suppressed lupus nephritis and prolonged survival in NZB/NZW mice . Final support for the potential use of irinotecan as a novel treatment strategy for SLE patients comes from our findings showing that irinotecan, even at a low dose, does not result in profound immunosuppression [20, 21, 22]. Instead, we presented evidence that alterations in DNA relaxation and subsequent changes in binding of anti-dsDNA antibodies may account for irinotecan-mediated effects in SLE [21, 22].
Are these data sufficient enough to legitimize first clinical trials treating lupus patients with irinotecan? Yes, we think so. However, to provide additional support in favor of clinical testing of irinotecan in human SLE, we introduced the MRL/lpr mouse model which is genetically different from NZB/NZW . MRL/lpr mice were treated with either high-dose or low-dose irinotecan. As a result, high-dose irinotecan clearly suppressed the development of lupus-associated glomerulonephritis and prevented the onset of skin lesions. When applying low-dose irinotecan to MRL/lpr mice, the results were more difficult to interpret. Undoubtedly, until week 20, low-dose irinotecan almost completely inhibited the onset of proteinuria, a time point when most of the MRL/lpr mice-utilizing studies have finished [42, 43, 44, 45]. Skin lesions were prevented by low-dose irinotecan at least until week 18, which was confirmed through histopathology assessed by a blinded pathologist. After week 18, an increasing number of mice from the group treated with low-dose irinotecan had to be sacrificed due to a skin score ≥4. The remaining mice of this group were suppressed for proteinuria, suggesting that low-dose irinotecan was more efficient at inhibiting lupus nephritis than skin lesions. However, it is very likely that intermediate dosages of irinotecan exist that are considerably lower than the doses used for chemotherapy, but which are still sufficient to suppress both lupus nephritis and skin lesions in MRL/lpr mice. This needs to be clarified in upcoming experiments.
Further investigation is also warranted for the finding that topo I-mediated DNA relaxation increased binding of anti-dsDNA IgG but not binding of anti-dsDNA IgM. Notably, anti-dsDNA IgM is known to have a protective function in SLE [46, 47, 48, 49]. However, our previous results showing that DNA relaxation and anti-dsDNA IgG binding can be inhibited by topo I inhibitors explain how irinotecan executes its protective function in SLE. Without the use of a topo I inhibitor, circulating DNA might be relaxed allowing increased binding of anti-dsDNA IgG followed by inflammatory disease. In contrast, when applying a topo I inhibitor, circulating DNA might be less relaxed and, subsequently, binding of anti-dsDNA IgG will be reduced. Then, assuming that binding of anti-dsDNA IgM is independent of DNA relaxation, more anti-dsDNA IgM with its lupus-protective function would be able to bind to circulating DNA. However, such a mechanism is very speculative thus far and needs to be preferentially shown in vivo.
Summarizing our results, inhibition of topo I is highly proficient at suppressing lupus nephritis and other symptoms of lupus-like disease in two different mouse models of spontaneous SLE. Although the mechanism is not fully understood yet, it seems that topo I inhibition represents a specific and targeted therapy for SLE beyond immunosuppression. Therefore, first clinical trials applying low-dose irinotecan to human SLE patients are urgently required.
The topo I inhibitor irinotecan suppressed both lupus nephritis and lupus-like skin lesions in MRL/lpr mice. Immunosuppression was not significantly involved in irinotecan-mediated suppression of lupus-like disease in MRL/lpr mice. As an alternative mechanism for irinotecan-mediated effects on lupus-like disease, our data propose altered binding of pathogenic anti-dsDNA IgG as a result of changes in DNA relaxation.
In view of the highly conserved nature of topo I, and considering the application of extremely low dosages of the topo I inhibitor as well as the lack of immunosuppression, inhibition of topo I might be developed as the first targeted therapy for human SLE.
This work was supported by the Swiss National Science Foundation (#310030_140964, grant to SF) and by the non-profit organization LUNIRI (https://www.luniri.com).
AK and SF designed the study, analyzed and interpreted the data, and wrote the manuscript. AK, SRH, and MK performed the experiments. SRH, MK, MH, and RAS made substantial contributions to data analysis, data interpretation, and drafting of the manuscript. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
Ethics approval and consent to participate
All experimental protocols were approved by the Kantonale Tierversuchskommission (Berne, Switzerland).
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