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Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. ROLE OF THE STUDY SPONSOR
  8. REFERENCES

Objective

Lupus nephritis depends on autoantibody deposition and activation of multiple immune cell types that promote kidney inflammation, including lymphocytes and monocyte/macrophages. Laquinimod, currently in clinical trials for multiple sclerosis and lupus nephritis, reduces infiltration of inflammatory cells into the spinal cord in experimental autoimmune encephalomyelitis. Activated monocyte/macrophages infiltrate the kidneys during nephritis in systemic lupus erythematosus (SLE). We undertook this study to determine whether using laquinimod to reduce monocyte/macrophage-driven tissue damage as well as to alter lymphocytes in SLE nephritis could have greater therapeutic benefit than current treatments that primarily affect lymphocytes, such as mycophenolate mofetil (MMF).

Methods

To test laquinimod efficacy, we used the (NZB × NZW)F1 mouse model of SLE, in which disease manifests as nephritis. Preventive and therapeutic studies were performed to determine whether laquinimod could prevent or delay nephritis, as measured by proteinuria, serum creatinine, survival, and renal pathology. Spleen and kidney leukocyte populations and suppression assays were analyzed by flow cytometry.

Results

Laquinimod prevented or delayed lupus manifestations at levels equal to or better than MMF. Laquinimod treatment was associated with reduced numbers of monocyte/macrophages, dendritic cells, and lymphocytes, as well as with induction of myeloid-derived suppressor cells in spleens and kidneys. Laquinimod suppressed macrophage-secreted tumor necrosis factor α and induced production of interleukin-10 (IL-10). In addition, laquinimod suppressed interferon-γ and IL-17 production by lymphocytes and down-regulated expression of activation/costimulatory markers on antigen-presenting cells.

Conclusion

The effects of laquinimod on myeloid and lymphoid cells may contribute to improvements in (NZB × NZW)F1 mouse survival, proteinuria, and glomerulonephritis. Future development of laquinimod as a therapeutic agent for lupus nephritis is promising.

Although glomerular inflammation in murine and human lupus nephritis is initiated by deposition of complement-fixing Ig, including IgG anti-DNA, both inflammation and damage are perpetuated by activation of multiple additional pathways ([1, 2]). Glucocorticoid therapy suppresses many of these pathways and is broadly antiinflammatory and immunosuppressive. However, it is not curative and is associated with many undesirable side effects. Therefore, it is now standard to treat lupus nephritis in humans with additional agents, including antimalarials (which suppress antigen presentation and Toll-like receptor [TLR] activation of dendritic cells) plus immunosuppressive drugs ([3]). Current standard of care often includes mycophenolate mofetil (MMF) or cyclophosphamide, which primarily target lymphocytes. Since none of these treatments is curative and many patients do not respond fully or have sustained improvement, there is an unmet need for newer approaches.

Renal tissue–fixed macrophages are activated early in the course of lupus nephritis and contribute to tissue damage, as do infiltrating monocyte/macrophages, lymphocytes, and neutrophils ([4]). Expansion of the resident macrophage population occurs in many forms of glomerulonephritis, including human lupus nephritis, and is associated with renal injury ([5]). In addition, kidney disease is attenuated if monocyte/macrophages are not recruited, such as in MRL/lpr systemic lupus erythematosus (SLE)–prone mice, which are deficient in macrophage migration inhibitory factor ([6]). Therefore, an intervention that targets monocyte/macrophages plus lymphocytes might be advantageous.

Laquinimod (5-chloro-N-ethyl-4-hydroxy-1-methyl- 2-oxo-N-phenyl-1,2-dihydroquinoline-3-carboxamide) is an immunomodulatory drug that has altered both lymphocytes and monocyte/macrophages in murine experimental autoimmune encephalomyelitis (EAE) ([7, 8]). It has been used successfully in clinical trials in patients with multiple sclerosis (MS), with a mild adverse events profile ([9]). In addition, laquinimod is currently in clinical trials for treating lupus nephritis. Therefore, we reasoned that laquinimod might be equivalent to or better than MMF in treating murine lupus nephritis.

In the present study, first-generation female (NZB × NZW)F1 (NZB/NZW) mice, which develop lupus nephritis, were treated orally with water, MMF, or laquinimod. Some mice were treated before clinical evidence of disease; others were treated after they were IgG anti-DNA positive or after they had heavy proteinuria. Laquinimod was as effective as MMF, either in preventing the onset of disease or in reducing renal disease in animals with advanced nephritis. Furthermore, while both MMF and laquinimod decreased lymphocyte numbers in the kidneys, only laquinimod reduced the numbers of renal monocyte/macrophages. In addition, laquinimod induced significantly higher proportions of both myeloid-derived suppressor cells (MDSCs) and antiinflammatory type II monocyte/macrophages and reduced proinflammatory type I monocyte/macrophages, interferon-γ (IFNγ)+CD4+ and interleukin-17A (IL-17A)+CD4–CD8– cells and serum IL-17A levels. These results suggest that laquinimod has the potential to target both myeloid and lymphoid cells as well as to delay or prevent human lupus nephritis.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. ROLE OF THE STUDY SPONSOR
  8. REFERENCES

Mice

Female NZB (H-2d/d), NZW (H-2z/z), and NZB/NZW (H-2d/z) mice were purchased from The Jackson Laboratory. Mice were treated in accordance with the guidelines of the University of California, Los Angeles, Animal Research Committee, an institution accredited by the Association for Assessment and Accreditation of Laboratory Animal Care.

Medication treatment

Preventive group

Eleven-week old (prenephritic) NZB/NZW mice were treated orally 3 times a week with water, 1 mg/kg (low-dose) laquinimod (Teva Pharmaceuticals), or 25 mg/kg (high-dose) laquinimod or were treated 5 times a week with 30 mg/kg (low-dose) MMF or 100 mg/kg (high-dose) MMF.

Therapeutic groups

The low-proteinuria group consisted of mice with IgG anti–double-stranded DNA (anti-dsDNA) detectable in serum and proteinuria ≤100 mg/dl, and the high-proteinuria group had proteinuria ≥300 mg/dl. Mice in both therapeutic groups were treated with water, 25 mg/kg laquinimod 3 times a week, or 100 mg/kg MMF 5 times a week. Results from the preventive group showed that these doses of laquinimod and MMF were more effective than the lower doses.

Reagents

Fluorochrome-conjugated antibodies to CD11b, Ly-6G, Ly-6C, Gr-1 (RB6-8C5), CD11c, CD3, CD4, CD8, CD19, FoxP3, IFNγ, arginase 1, IL-10, IL-12/IL-23, transforming growth factor β, tumor necrosis factor α (TNFα), and IL-17A used in flow cytometry experiments were from eBioscience, BioLegend, or BD Biosciences. Cell cultures were performed in RPMI 1640 (Cellgro) supplemented with l-glutamine (2 mM), penicillin (100 units/ml), streptomycin (0.1 mg/ml), 2-mercaptoethanol (Gibco), and 2% or 10% (volume/volume) fetal bovine serum (FBS). Enzyme-linked immunosorbent assay (ELISA) for IL-17A in mouse serum was performed as described by the manufacturer (BioLegend).

Cell isolation, proliferation, and cytokines

Single-cell suspensions of splenocytes or kidney cells were prepared using cell strainers (Fisher), followed by red blood cell lysis. Kidney lymphocytes were separated from connective tissue by centrifugation (15 minutes at 400g) in 30% Percoll (GE Healthcare). CD4+CD25– cells from spleen and kidney were isolated using a CD4+CD25+ regulatory T cell isolation kit according to the protocol of the manufacturer (Miltenyi Biotec). CD11b+ cells were isolated using an EasySep mouse CD11b positive selection kit (StemCell Technologies) and were surface stained for Ly-6C and Ly-6G prior to sorting MDSCs using a FACSAria II (BD Biosciences). Populations were >95% pure by fluorescence-activated cell sorter analysis.

CD4+CD25– cells were labeled with 5 μM 5,6-carboxyfluorescein succinimidyl ester and stimulated with Dynabeads mouse T cell activator CD3/CD28 (Invitrogen). Stimulated CD4+CD25– cells (4 × 104) were cocultured with monocytic MDSCs (CD11b+Ly-6C+Ly-6G–) or granulocytic MDSCs (CD11b+Ly-6C+Ly-6G+) at a 1:1 ratio. Proliferation of CD4+ cells was assayed by flow cytometry after 4 days of incubation at 37°C in 5% CO2.

For intracellular cytokines, whole splenocytes were stimulated for 24 hours with 1 μg/ml lipopolysaccharide (Sigma), 10 μg/ml imiquimod (a TLR-7 agonist), or 10 μg/ml oligonucleotide (a TLR-9 agonist) in 2% FBS/RPMI 1640, with the last 12 hours in the presence of monensin (BioLegend), or were stimulated for 5 hours with 50 ng/ml phorbol 12-myristate 13-acetate (PMA) plus 500 ng/ml ionomycin in the presence of monensin. Intracellular expression of cytokines in CD4+CD8– or CD4–CD8– double-negative lymphocytes and macrophages (CD11b+Ly-6C–Ly-6G–) was analyzed by intracellular flow cytometry.

Clinical analysis of murine SLE for proteinuria, creatinine, and anti-dsDNA antibodies

Proteinuria was evaluated weekly using Albustix (Siemens), serum creatinine using a commercial kit (Arbor Assays), and IgG anti-dsDNA antibodies by ELISA as described previously ([10]).

Flow cytometry

Single-cell suspensions from spleen or kidneys were stained for extracellular markers for 20 minutes at 4°C in phosphate buffered saline (PBS)/1% FBS. Intracellular staining for FoxP3 and cytokines was performed using a FoxP3 Fixation/Permeabilization kit according to the protocol of the manufacturer (eBioscience). Cells were acquired on an LSRII flow cytometer (BD Biosciences), and data were analyzed using FlowJo software (Tree Star).

Immunofluorescence staining and histology

Kidney cryosections (4 μm) were stained with fluorescein isothiocyanate–conjugated goat anti-mouse IgG (Sigma) or rat anti-mouse C3 (Solulink) in 2% normal goat or rat serum and 2% bovine serum albumin/PBS for 2 hours at room temperature. Images were acquired using a Nikon Eclipse TE2000-U microscope and MetaMorph software, version 6.3 (Molecular Devices). The immunostained area of tissue sections was quantified using ImageJ software (http://rsb.info.nih.gov/ij/index.html).

For histologic analysis, 10% formalin–fixed, paraffin-embedded kidney sections (4 μm) were stained with hematoxylin and eosin, periodic acid–Schiff, and methenamine–silver (Jones) stain. Acute inflammatory features (endocapillary hypercellularity, mesangial hypercellularity, cellular necrosis, cellular crescents, interstitial inflammation), chronic features (global glomerulosclerosis, focal segmental glomerulosclerosis [FSGS]), and interstitial fibrosis or tubular atrophy were analyzed by an investigator (MFP-D) who was blinded to the treatment groups. Glomeruli (25 per section) of at least 10 mice per group were scored as 0 (lesions absent), 1 (lesions involving up to 25% of the glomeruli for the component evaluated), 2 (lesions in 26–50% of the glomeruli), or 3 (lesions in >50% of the glomeruli). Histologic scores are reported as the mean ± SEM of each feature per group.

Statistical analysis

Statistical analyses were performed using GraphPad Prism software, version 4. Comparisons between 2 groups were performed by 2-tailed t-test. Tests between more than 2 groups were performed by one-way analysis of variance (ANOVA). In cases where repeated measures were assessed, two-way ANOVAs were used. Tukey's multiple comparison test (one-way ANOVA) and Bonferroni post test (two-way ANOVA) were used in testing between pairs of groups. Survival was analyzed by log rank test, and statistical significance was adjusted to account for multiple comparisons using the Bonferroni method. P values less than 0.05 were considered significant. With a sample size of 6 mice in each group, the minimally detectable (with 80% power) effect size is 1.33, assuming a 2-group t-test with a 2-sided alpha level of 0.05. The magnitude of the minimally detectable effect was smaller than that observed in pilot studies, suggesting that this sample size was sufficient.

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. ROLE OF THE STUDY SPONSOR
  8. REFERENCES

Laquinimod suppresses murine lupus nephritis in a preventive study

The study had 3 treatment arms. The preventive study tested whether laquinimod delayed onset of disease by treating young, prediseased mice. The therapeutic low-proteinuria study tested whether laquinimod treatment of mice with IgG anti-dsDNA but with proteinuria ≤100 mg/dl could delay kidney damage. The therapeutic high-proteinuria study tested whether laquinimod treatment could reverse disease in mice with anti-dsDNA and heavy proteinuria (≥300 mg/dl).

In the preventive study, anti-dsDNA serum levels were significantly lower in laquinimod-treated mice than in vehicle-treated animals after 13–18 weeks of treatment and were similar to levels in mice treated with 100 mg/kg MMF (data not shown). Survival was significantly better in mice receiving low doses of laquinimod and MMF than in vehicle-treated mice (Figure 1A). Both doses of laquinimod completely prevented proteinuria over the 18-week study period (Figure 1B). MMF at 100 mg/kg prevented proteinuria in a manner similar to both laquinimod doses, but MMF at 30 mg/kg did not (Figure 1B). A rise in serum creatinine levels was significantly delayed only by treatment with 25 mg/kg laquinimod after 18 weeks of treatment (Figure 1C).

image

Figure 1. Laquinimod prevents lupus nephritis in preventively treated (NZB × NZW)F1 mice. Young mice (age 12 weeks) without established disease were treated orally with vehicle, laquinimod (1 mg/kg [LAQ 1] or 25 mg/kg [LAQ 25]), or mycophenolate mofetil (MMF) (30 mg/kg [MMF 30] or 100 mg/kg [MMF 100]). Shown are the percent survival (A), percent of mice with proteinuria ≥300 mg/dl (B), serum creatinine levels (C), periodic acid–Schiff staining (original magnification × 400) (D), and histologic scores of kidney sections (E). In C and E, values are the mean ± SEM and are representative of 3 independent experiments with similar results. ∗∗ = P < 0.01; ∗∗∗ = P < 0.001 versus vehicle-treated group. # = P < 0.01. ANOVA = analysis of variance; GS = glomerulosclerosis; FSGS = focal segmental glomerulosclerosis; IF/TA = interstitial fibrosis or tubular atrophy.

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Because NZB/NZW mice develop renal failure with IgG and C3 deposition as well as acute and chronic pathologic changes ([11]), we examined renal specimens from 35-week-old control and treated mice. Both laquinimod and MMF reduced deposition of IgG and C3 in glomeruli compared to that in control mouse kidneys (further data available upon request from the corresponding author). In addition, compared with vehicle, treatment with both drugs significantly prevented development of acute pathologic features such as endocapillary and mesangial hypercellularity, and chronic features such as FSGS (Figures 1D and E). These data suggest that treatment with either laquinimod or MMF delays the development of lupus nephritis. Laquinimod appears to be at least as effective as MMF in delaying kidney damage in murine SLE. In summary, the preventive experiments illustrated that laquinimod treatment at both doses was as effective as 100 mg/kg MMF with regard to anti-dsDNA, survival, and proteinuria, but only laquinimod at 25 mg/kg prevented a rise in the serum creatinine level. Thus, we used the high doses of laquinimod (25 mg/kg) and MMF (100 mg/kg) for the therapeutic arms of the study.

Therapeutic treatment with laquinimod delays disease progression in NZB/NZW mice

In the therapeutic low-proteinuria study, anti-dsDNA levels were not significantly decreased in 25 mg/kg laquinimod–treated or 100 mg/kg MMF–treated mice (data not shown). However, laquinimod and MMF significantly prolonged survival (Figure 2A) and prevented increases in proteinuria (Figure 2B) and serum creatinine (Figure 2C). Although IgG and C3 deposits in the glomeruli from both laquinimod- and MMF-treated mice were not significantly different from those in vehicle-treated mice (further data available upon request from the corresponding author), both treatments were equally effective in reducing endocapillary hypercellularity and FSGS in mice with anti-dsDNA and low proteinuria (Figures 2D and E). Necrosis and crescent formation were uncommon in all groups (data not shown).

image

Figure 2. Laquinimod ameliorates lupus nephritis in therapeutically treated (NZB × NZW)F1 mice with anti–double-stranded DNA and low proteinuria. Mice were treated orally with vehicle, laquinimod, or MMF. Shown are the percent survival (A), percent of mice with proteinuria ≥300 mg/dl (B), serum creatinine levels (C), periodic acid–Schiff staining (original magnification × 400) (D), and histologic scores of kidney sections (E). In C and E, values are the mean ± SEM and are representative of 4 independent experiments with similar results. ∗ = P < 0.05; ∗∗ = P < 0.01; ∗∗∗ = P < 0.001 versus vehicle-treated group. NS = not significant (see Figure 1 for other definitions).

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In the therapeutic high-proteinuria study, 25 mg/kg laquinimod treatment significantly increased survival, and this was not observed in mice receiving 100 mg/kg MMF (Figure 3A). Similarly, laquinimod treatment significantly reduced proteinuria to levels below 300 mg/dl in 50% of animals compared to vehicle-treated controls (Figure 3B). Although ∼25% of MMF-treated animals also experienced a drop in proteinuria below 300 mg/dl, the difference from controls was not significant (Figure 3B). Both treatments prevented a rise in serum creatinine levels (Figure 3C), but deposition of IgG and C3 in the glomeruli was not different from that in controls (data not shown). In contrast, endocapillary hypercellularity, FSGS, and interstitial fibrosis or tubular atrophy were significantly decreased in laquinimod-treated mice (Figures 3D and E). MMF treatment also reduced FSGS and interstitial fibrosis or tubular atrophy, but not endocapillary hypercellularity (Figures 3D and E). Similar to findings in the therapeutic low-proteinuria study, necrosis and crescent formation were uncommon events in all treatment groups. Together, these data show that laquinimod suppresses clinical and histologic manifestations of murine lupus nephritis and increases survival in therapeutic treatment regimens at levels equal to or better than MMF.

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Figure 3. Laquinimod ameliorates lupus nephritis in therapeutically treated (NZB × NZW)F1 mice with high proteinuria. Mice were treated orally with vehicle, laquinimod, or MMF. Shown are the percent survival (A), percent of mice with proteinuria ≥300 mg/dl (B), serum creatinine levels (C), periodic acid–Schiff staining (original magnification × 400) (D), and histologic scores of kidney sections (E). In C and E, values are the mean ± SEM and are representative of 4 independent experiments with similar results. ∗ = P < 0.05; ∗∗ = P < 0.01; ∗∗∗ = P < 0.001 versus vehicle-treated group. # = P < 0.01. NS = not significant (see Figure 1 for other definitions).

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Laquinimod treatment affects both myeloid and lymphoid immune cells

Previous studies have shown that laquinimod is an immunomodulator that affects various leukocyte populations but preferentially targets antigen-presenting cells (APCs) in EAE models ([7, 8, 12]). We investigated the effect of laquinimod on different leukocyte subsets in the kidney and spleen of NZB/NZW mice in the preventive and therapeutic low-proteinuria groups after 25 and 10 weeks of treatment, respectively (this was not done in the therapeutic high-proteinuria group because control animals did not survive long enough to be compared with treated animals).

In both spleen and kidney, there was a significant increase in the frequency of CD11b+Ly-6C+Ly-6G+ cells in mice treated with laquinimod in the preventive and therapeutic low-proteinuria arms compared with the frequency of these cells in MMF- and vehicle-treated animals (Figure 4A and Table 1) (further data available upon request from the corresponding author). CD11b+Ly-6C+Ly-6G– cells were increased in the kidneys of laquinimod-treated mice (Figure 4B and Table 1) (further data available upon request from the corresponding author). In contrast, a significant decrease in CD11b+Ly-6C–Ly-6G– (CD11b+MDSC–) monocyte/macrophages was observed in both spleen and kidney of laquinimod-treated mice (Figure 4C and Table 1) (further data available upon request from the corresponding author). The numbers of CD4+, CD8+, and CD19+ cells were significantly lower in the kidneys of laquinimod-treated mice in both the preventive and therapeutic low-proteinuria arms compared with numbers of these cells in controls (Table 1). These cell numbers were also significantly decreased by MMF treatment in the therapeutic low-proteinuria group (Table 1). The numbers of CD11c+CD11b+ myeloid dendritic cells were significantly decreased in the kidneys (preventive and therapeutic low-proteinuria arms) and spleens (preventive arm only) of laquinimod-treated animals compared with numbers of these cells in controls (Table 1) (further data available upon request from the corresponding author). Therefore, laquinimod treatment affects cells from myeloid and lymphoid compartments in NZB/NZW mice.

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Figure 4. Induction of myeloid-derived suppressor cells (MDSCs) and inhibition of monocyte/macrophages after laquinimod treatment. Two groups of (NZB × NZW)F1 mice were treated orally with vehicle, laquinimod, or MMF. A preventive treatment group of prenephritic mice (Prev) was treated before appearance of anti–double-stranded DNA (anti-dsDNA), and cells from spleen and kidney in these mice were investigated by fluorescence-activated cell sorting (FACS) 25 weeks after treatment was initiated. A group of mice with low proteinuria (PUlo) had IgG anti-dsDNA detectable in serum and proteinuria ≤100 mg/dl, and cells from spleen and kidney in these mice were investigated by FACS 10 weeks after treatment was initiated. AC, Graphs compare percentages of CD11b+Ly-6C+Ly-6G+ cells (A), CD11b+Ly-6C+Ly-6G– cells (B), and monocyte/macrophages (CD11b+Ly-6C–Ly-6G– cells) (C). D, Both FACS data and the bar graph show the percentage of proliferating CD4+ T cells (5,6-carboxyfluorescein succinimidyl ester [CFSE] dilution) following culture in the presence (at a 1:1 ratio) or absence of either granulocyte-like MDSCs (gr-MDSC) or monocyte-like MDSCs (mo-MDSC) sorted from spleens of laquinimod-, MMF-, or vehicle-treated mice. Values are the mean ± SEM and are representative of 3 independent experiments with similar results. ∗ = P < 0.05; ∗∗ = P < 0.01; ∗∗∗ = P < 0.001 versus vehicle-treated group, by one-way ANOVA. # = P < 0.05; ## = P < 0.001. Med = medium (see Figure 1 for other definitions).

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Table 1. Leukocyte subsets present in mouse kidney after preventive and therapeutic treatment*
 Preventive treatmentTherapeutic treatment
Leukocyte subsetVehicleLaquinimodMMFVehicleLaquinimodMMF
  1. We investigated the effect of laquinimod on different leukocyte subsets in the kidney of (NZB × NZW)F1 mice in the preventive and therapeutic low-proteinuria groups after 25 weeks of treatment (n = 5–10 mice per group) and 10 weeks of treatment (n = 4–10 mice per group), respectively. Values are the mean ± SEM frequency of cells. CD11b+myeloid-derived suppressor cell (MDSC)–negative = monocyte/macrophages; CD11c+CD11b+ = myeloid dendritic cells (DCs); CD11c+CD11b– = lymphoid DCs; CD11b+Ly-6C+Ly-6G– = monocyte-like MDSCs; CD11b+Ly-6ClowLy-6G+ = granulocyte-like MDSCs.

  2. a

    P < 0.001 versus vehicle and versus mycophenolate mofetil (MMF), by one-way analysis of variance (ANOVA).

  3. b

    P < 0.01 versus vehicle and P < 0.05 versus MMF, by one-way ANOVA.

  4. c

    P < 0.001 versus vehicle, by one-way ANOVA.

  5. d

    P < 0.01 versus vehicle and versus MMF, by one-way ANOVA.

CD11b+MDSC−44.7 ± 0.731.0 ± 1.8a49.8 ± 0.744.7 ± 0.728.4 ± 0.9a45.7 ± 0.9
CD11c+CD11b+1.4 ± 0.20.7 ± 0.1b1.0 ± 0.11.5 ± 0.10.7 ± 0.1c0.6 ± 0.0c
CD11c+CD11b−3.8 ± 0.33.8 ± 0.24.4 ± 0.31.8 ± 0.31.2 ± 0.11.3 ± 0.1
CD11b+Ly-6C+Ly-6G−21.0 ± 0.944.4 ± 1.5a22.9 ± 0.821.5 ± 0.741.2 ± 0.4a22.4 ± 0.3
CD11b+Ly-6ClowLy-6G+1.2 ± 0.27.9 ± 1.9d1.6 ± 0.21.3 ± 0.113.6 ± 1.2a1.9 ± 0.3
CD4+18.3 ± 0.57.0 ± 0.5a19.4 ± 0.44.2 ± 0.12.8 ± 0.3a1.0 ± 0.1c
CD8+9.3 ± 0.34.2 ± 0.2a8.8 ± 0.113.4 ± 0.82.7 ± 0.2d6.6 ± 0.2c
CD19+5.7 ± 0.14.0 ± 0.5d2.3 ± 0.2c12.2 ± 0.22.7 ± 0.2a7.2 ± 0.4c

Laquinimod induces expansion of MDSCs

Previous studies have shown that cells coexpressing CD11b, Ly-6G, and/or Ly-6C are endowed with suppressive activity and are called myeloid-derived suppressor cells ([13-15]). MDSCs can be subdivided into 2 distinct subpopulations with monocytic or granulocytic morphology and are defined as CD11b+Ly-6C+Ly-6G− (monocyte-like MDSCs) or CD11b+Ly-6ClowLy-6G+ (granulocyte-like MDSCs) ([13, 15]). We observed increased frequencies of both subpopulations in laquinimod-treated animals, as discussed above. We further investigated their suppressive function in cocultures with stimulated CD4+ T cells and found that both types of MDSCs purified from laquinimod-treated animals strongly suppressed T cell proliferation (Figure 4D). The effects of laquinimod on the generation of MDSCs did not appear to affect functionality of MDSCs, because both types of MDSCs sorted from MMF- and vehicle-treated mice displayed comparable suppressive activity. Also, production of arginase and reactive oxygen species, 2 of the major factors involved in suppression by MDSCs ([16]), were not affected by laquinimod treatment (data not shown). These data suggest that laquinimod might be effective in treating lupus nephritis in NZB/NZW mice due in part to its ability to induce MDSCs.

Laquinimod promotes a cytokine shift to an antiinflammatory profile in NZB/NZW mice

Recent evidence suggests that laquinimod induces antiinflammatory type II monocytes in EAE ([8, 12]). Therefore, we investigated cytokine production by splenic monocyte/macrophages (CD11b+Ly-6C–Ly-6G–) from laquinimod- or vehicle-treated mice after in vitro stimulation with TLR agonists. We found that monocyte/macrophages from laquinimod-treated mice expressed significantly more IL-10 and less TNFα than monocyte/macrophages from the vehicle-treated group (Figure 5A). A detailed surface phenotypic analysis of monocyte/macrophages from laquinimod- and vehicle-treated animals revealed decreased ex vivo and/or in vitro expression of the activation/costimulatory molecules class II major histocompatibility complex (MHC), CD86, CD80, and CD40 in monocyte/macrophages from laquinimod-treated animals (Figure 5B). Down-regulation of activation/costimulatory molecules was also observed on B cells and dendritic cells (data not shown). High expression of IL-10, low expression of TNFα, and down-regulation of CD80 and CD86 are characteristics of antiinflammatory type II macrophages ([17, 18]). Our data suggest that laquinimod promotes switching of monocyte/macrophages from proinflammatory (type I) to antiinflammatory (type II).

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Figure 5. Laquinimod suppresses proinflammatory cytokines, promotes interleukin-10 (IL-10) production, and down-regulates activation/costimulatory molecules. Spleens were removed 7 weeks after initiation of laquinimod treatment in animals in the low-proteinuria group (see Materials and Methods). Splenocytes were stimulated with agonists of Toll-like receptor 4 (TLR-4) (lipopolysaccharide [LPS]), TLR-7 (imiquimod), or TLR-9 (oligonucleotide), or in the presence of monensin (PIM). A, Left, Fluorescence-activated cell sorting plots are representative of 1 experiment showing intracellular production of IL-10 and tumor necrosis factor α (TNFα) by monocyte/macrophages. Right, Bars show the mean ± SEM of cytokine production (n = 6 mice per group). ∗ = P < 0.05; ∗∗∗ = P < 0.001 versus vehicle (veh)–treated group, by two-way ANOVA. B, Ex vivo and in vitro (with 1 μg/ml LPS for 24 hours) expression of activation/costimulatory molecules on monocyte/macrophages is down-regulated by laquinimod treatment. Bars show the mean ± SD mean fluorescence intensity (MFI) from 3 independent experiments with similar results (n = 6 mice per group). ∗ = P < 0.05; ∗∗ = P < 0.01; ∗∗∗ = P < 0.001 versus vehicle-treated group, by t-test. C, Left, Interferon-γ (IFNγ) and IL-17A production was measured in cells in the lymphocyte gate, as described for the macrophage gate in A. Right, Bars show the mean ± SEM cytokine production. ∗∗ = P < 0.01 versus vehicle-treated group, by two-way ANOVA. # = P < 0.05. D, Serum IL-17A levels were measured before and 14 and 20 weeks after initiation of laquinimod treatment in animals in the low-proteinuria group. Bars show the mean ± SEM (n = 6 mice per group). ∗∗ = P < 0.01; ∗∗∗ = P < 0.001 versus vehicle-treated group, by two-way ANOVA. Med = medium; MHC-II = class II major histocompatibility complex; DN = double-negative (see Figure 1 for other definitions).

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We next examined intracellular expression of IFNγ in splenic lymphocytes after in vitro stimulation with PMA plus ionomycin in the presence of monensin. CD4+ splenocytes from laquinimod-treated mice made significantly less IFNγ than did CD4+ cells from vehicle-treated mice (Figure 5C). In addition, because laquinimod down-regulates IL-17 production ([7]) and Th17 cells ([8]), and both play important roles in the pathogenesis of lupus nephritis ([19, 20]), we examined in vitro expression of intracellular IL-17 after stimulation in the presence of monensin. In contrast to CD4+ cells, expression of IL-17A in CD4–CD8– double-negative lymphocytes was significantly lower in laquinimod-treated animals than in vehicle-treated animals (Figure 5C). This is consistent with the finding that double-negative T cells expressed significantly more IL-17A than did CD4+ T cells in lupus-prone mice ([20]). Serum IL-17 levels were also lower in laquinimod-treated mice over the course of treatment (Figure 5D). These results suggest that putative mechanisms of laquinimod in lupus nephritis involve inhibition of the proinflammatory cytokines IFNγ and IL-17.

DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. ROLE OF THE STUDY SPONSOR
  8. REFERENCES

In this study, we explored the immunomodulatory properties of laquinimod in murine lupus nephritis. Our data indicate that laquinimod treatment, whether preventive or therapeutic, led to prevention or improvement in established proteinuria, decreased rise in serum creatinine, and improved survival. In mice with active advanced disease as defined by the presence of anti-dsDNA and proteinuria ≥300 mg/dl, laquinimod significantly improved survival and proteinuria, which MMF treatment failed to do. In addition, inhibition of clinical disease by laquinimod was associated with an increased frequency of MDSCs, a switch of monocyte/macrophages from proinflammatory type I to antiinflammatory type II, down-regulation of activation/costimulatory molecules on APCs, and reduced numbers of IFNγ- and IL-17–producing cells. The numbers of MDSCs and monocyte/macrophages were unchanged in MMF-treated mice. Notably, modulation of these cell types in laquinimod-treated mice was observed not only in the spleen, but also in the kidney, the target tissue in lupus nephritis, and may represent a distinct advantage of laquinimod therapy over commonly used therapeutics when used in human SLE.

Results from a clinical trial of laquinimod in human SLE nephritis were recently reported at the 2013 Congress of the European League Against Rheumatism (EULAR 2013) ([21]). Lower levels of proteinuria and improved complete renal response trended toward significance in patients who added laquinimod to a treatment regimen that also included MMF and prednisone, with no differences in reported adverse events. These results suggest that laquinimod could be effective in treating human lupus nephritis.

The major cause of death in NZB/NZW mice is glomerulonephritis ([22]). Thus, one would expect that improved survival and prevention of rises in creatinine levels in laquinimod-treated animals would correlate with protection from kidney damage. Histologic analysis of preventive and therapeutic (both low-proteinuria and high-proteinuria arms) treatments demonstrated that laquinimod significantly reduced acute and chronic features of nephritis, including endocapillary hypercellularity, FSGS, and interstitial fibrosis or tubular atrophy. Preventive treatment was also effective in preventing IgG and C3 deposition in the glomeruli. However, neither laquinimod nor MMF prevented glomeruli deposits when administered after disease onset. This is consistent with previous studies that show unchanged deposition of antibodies in the glomeruli despite improvement of clinical disease after Ad-BAFF-R-Ig treatment ([23]).

In previous studies, it was observed that a beneficial effect of laquinimod in EAE models was associated with a cytokine shift from a proinflammatory to an antiinflammatory monocyte/macrophage phenotype ([7, 12, 24]). Laquinimod treatment induced antiinflammatory (type II) monocytes and reduced secretion of IFNγ and IL-17 in EAE ([8, 12, 25]). Similarly, we show here that laquinimod induces antiinflammatory (type II) and reduces proinflammatory (type I) monocyte/macrophages in NZB/NZW mice. Inflammatory/antiinflammatory functions of monocyte/macrophages exist along a spectrum, and we have simplified our studies by defining proinflammatory type I monocyte/macrophages as secreting TNFα and displaying high quantities of class II MHC, CD86, and CD80 as defined elsewhere ([17, 18]). Antiinflammatory type II monocyte/macrophages were defined as IL-10–secreting monocyte/macrophages with lower expression of the surface molecules mentioned above.

Laquinimod also reduced intracellular expression of IFNγ and IL-17A in NZB/NZW mice. Serum IL-17A levels were also significantly lower after laquinimod treatment in these lupus-prone animals. This is consistent with the finding that laquinimod suppresses Th17 cells/IL-17 in EAE ([7, 8]) and that IL-17+ cells play an important role in the development of lupus nephritis ([20]). Moreover, previous studies have suggested that laquinimod exerts beneficial effects in EAE through inhibition of leukocyte migration into the central nervous system ([7, 24, 26, 27]). Our data show that laquinimod also reduced the frequency of monocyte/macrophages, CD11c+CD11b+ dendritic cells, and CD4+, CD8+, and CD19+ cells in the kidneys. This suggests that suppression of disease severity by laquinimod in NZB/NZW mice might also be due to reduction in renal infiltration of effector cells that secrete inflammatory mediators and respond to immune complexes in the kidney.

Both preventive and therapeutic laquinimod treatment induced expansion of monocyte-like MDSCs and granulocyte-like MDSCs in NZB/NZW mice. MDSCs consist of a heterogeneous population of immature myeloid cells, immature granulocytes, monocyte/macrophages, dendritic cells, and myeloid progenitor cells ([16]). In mice, they are defined as CD11b+Gr-1+ cells. Gr-1 is an antibody (RB6-8C5) that detects both Ly-6G, a molecule expressed on granulocytes ([28]), and Ly-6C, a molecule highly expressed on monocytes ([29, 30]). MDSCs were originally described more than 25 years ago in patients with cancer ([31]) and have become the focus of intense study by immunologists in recent years, after studies by Bronte and colleagues ([32]). MDSCs are believed to regulate immune response under many pathologic conditions, including infections ([33-35]), acute inflammation ([36]), and various autoimmune diseases such as encephalomyelitis ([37]), colitis ([38]), diabetes ([39]), and a murine model of rheumatoid arthritis ([40, 41]). In addition, MDSCs may directly influence Th17 cell differentiation in experimental autoimmune ([41, 42]) and tumor ([43]) systems. Our results suggest that the effect of laquinimod on MDSCs is most likely quantitative rather than qualitative, as MDSCs from control animals also suppressed T cell proliferation. Therefore, a therapeutic option that increases numbers of these regulatory cells may be of interest in lupus nephritis.

A phase III clinical trial of laquinimod in MS demonstrated that the adverse events profile of laquinimod is mild, so the drug might also be safe in humans with lupus nephritis. Laquinimod-treated MS patients had a 2.6-fold higher risk of elevated alanine aminotransferase levels, a 2.0-fold higher risk of abdominal pain, and a 1.8-fold higher risk of back pain ([9]). This is a much more favorable side effects profile than those of both MMF (gastrointestinal distress, infections, leukopenia) and roquinimex, a quinolone closely related to laquinimod that was pulled out of a clinical trial because of serious cardiovascular toxicities ([44]). A recent trial of laquinimod in human lupus nephritis has been fully enrolled, and preliminary results were presented at EULAR 2013, although results have not been published to date (www.clinicaltrials.gov, searched September 2013). It is noteworthy that the laquinimod dosage used in the studies reported here is similar to the dosage in the MS clinical trial when adjusting for body surface area differences between humans and mice ([45]). In addition, paquinimod, a quinoline related to laquinimod that differs at a single side chain (a chloride ion in laquinimod versus an ethyl group in paquinimod), also showed a favorable adverse events profile in human lupus in a recent phase Ib trial and was effective at treating a different murine SLE model ([46]).

Our findings suggest that laquinimod ameliorates murine lupus nephritis through multiple mechanisms. Numbers of proinflammatory T and B lymphocytes, dendritic cells, and monocyte/macrophages were reduced in the target tissue (kidney). In addition, a shift from proinflammatory type I to antiinflammatory type II monocyte/macrophages and induction of 2 types of MDSCs were observed after laquinimod treatment. Proinflammatory cytokines made by monocyte/macrophages (TNFα) and lymphocytes (IFNγ, IL-17A) were also reduced after treatment. Laquinimod was more effective than MMF with regard to survival and reduction of proteinuria in mice with advanced lupus nephritis. Thus, laquinimod is a promising immunomodulatory therapeutic agent for use in human SLE and could be useful in treating human lupus nephritis, for which it is currently in clinical development.

AUTHOR CONTRIBUTIONS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. ROLE OF THE STUDY SPONSOR
  8. REFERENCES

All authors were involved in drafting the article or revising it critically for important intellectual content, and all authors approved the final version to be published. Dr. Skaggs had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

Study conception and design. Lourenço, Wong, Hahn, Skaggs.

Acquisition of data. Lourenço, Wong, Hahn, Palma-Diaz, Skaggs.

Analysis and interpretation of data. Lourenço, Wong, Hahn, Palma-Diaz, Skaggs.

ROLE OF THE STUDY SPONSOR

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. ROLE OF THE STUDY SPONSOR
  8. REFERENCES

Teva Pharmaceuticals provided laquinimod and funding for this project but had no role in the study design or in the collection, analysis, or interpretation of the data, the writing of the manuscript, or the decision to submit the manuscript for publication. Publication of this article was not contingent upon approval by Teva Pharmaceuticals.

REFERENCES

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. ROLE OF THE STUDY SPONSOR
  8. REFERENCES
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