DEK-targeting DNA aptamers as therapeutics for inflammatory arthritis

Abstract

Novel therapeutics are required for improving the management of chronic inflammatory diseases. Aptamers are single-stranded RNA or DNA molecules that have recently shown utility in a clinical setting, as they can specifically neutralize biomedically relevant proteins, particularly cell surface and extracellular proteins. The nuclear chromatin protein DEK is a secreted chemoattractant that is abundant in the synovia of patients with juvenile idiopathic arthritis (JIA). Here, we show that DEK is crucial to the development of arthritis in mouse models, thus making it an appropriate target for aptamer-based therapy. Genetic depletion of DEK or treatment with DEK-targeted aptamers significantly reduces joint inflammation in vivo and greatly impairs the ability of neutrophils to form neutrophil extracellular traps (NETs). DEK is detected in spontaneously forming NETs from JIA patient synovial neutrophils, and DEK-targeted aptamers reduce NET formation. DEK is thus key to joint inflammation, and anti-DEK aptamers hold promise for the treatment of JIA and other types of arthritis.

Figure 1. Zymosan induction of joint inflammation is impaired in Dek-KO mice.

(a) WT and Dek-KO mice were injected on day 0 with PBS or zymosan. Circumferences were measured at time of injection (day 0), as well as at 24 h after injection. Mean values of the increase in knee circumference are shown at 24 h after injection. The difference in the circumference at 24 h between WT and Dek-KO mice was statistically significant (*P=0.0006), as determined by two-tailed, unpaired Student's t-test (error bars, s.e.m.). Results shown are for individual mice and the average of four WT versus four Dek-KO for PBS control and 20 WT versus 23 Dek-KO mice for 24 h after zymosan injection (results shown are from four independent experiments). (b) Representative hematoxylin and eosin staining of knee joint sections of three WT and three Dek-KO mice 24 h after zymosan injection are shown in the top panel. In the middle panel, joint sections were analysed for neutrophils by immunohistochemistry using the murine neutrophil surface marker Ly6G (in red) 24 h after intra-articular injection. Sections stained for cell nuclei with DAPI (blue) are shown in the lower panel. Magnification × 40 (scale bar 100 μm). (c) Arbitrary fluorescent intensity of Ly6G staining was analysed for three different fields of each section of six WT and five Dek-KO zymosan-injected knees (*P=0.0364), as determined by two-tailed, unpaired Student's t-test (error bars, s.e.m.). Mean fluorescent intensity of the whole section was determined by Image J. (d) Inflammatory cytokine profile of knee homogenates 24 h after zymosan injection, as detected by ELISA. Cytokine levels were normalized by protein concentration and were significantly lower in Dek-KO versus WT knee homogenates (error bars, s.e.m.). Results shown are average cytokine levels from 7 WT and 11 Dek-KO mice.

Figure 2. Zymosan induction of joint inflammation is blocked by DEK aptamers.

(a)WT mice were injected on day 0 with 5, 50 or 100 ng per knee of non-specific DNA aptamer controls or DEK-specific aptamer (DTA-64) prior to injection with PBS alone or zymosan alone as negative and positive controls, respectively. Knee circumferences were measured at the time of injection (day 0), as well as at 48 h following injection. (b) Mean values of increased knee circumferences are shown at 48 h after injection. Results shown are from 4–8 individual mice per group and from two independent experiments (n=53 mice). Differences in the knee circumference between mice receiving control aptamers versus DTA 64 aptamer (5–100 ng) were statistically significant (*P<0.032) as determined by two-tailed, unequal variance Student's t-test. (c) Representative sections from seven knees injected with 50 ng control aptamer versus seven knees injected with 50 ng DTA-64 stained by H&E at 48 h after injection. Arrowheads indicate inflammatory cell migration into the knee joint. Magnification × 10 (scale bar 200 μm). (d) Semi-quantitative scoring of blinded histological assessment from control aptamer-injected knees (n=8) or DTA-64-injected knees (n=8) at 50 ng aptamer per knee (**P=0.0056) as determined by two-tailed, unequal variance Student's t-test. Results are from two independent experiments (error bars, s.e.m.).

Figure 3. Anti-DEK aptamers reduce NET formation in zymosan-injected joints.

(a) Representative confocal microscopy images of permeabilized joint sections from knees injected with zymosan and control aptamer (n=3) or anti-DEK aptamer (n=3) stained for Ly6-G (green) or the NET marker citrullinated histone H3 (cit–H3) antibody (red). Magnification × 60 (scale bar 20 μm). Staining for cit-H3 is undetectable in the neutrophils in the DTA 64-injected knees, but is readily seen in the control aptamer-injected joints. The absence of cit-H3 in the DTA-64 injected-knees correlates with the loss of NETs. (Please note that permeabilization of the cells makes it difficult to detect NET structures; please see NET staining in the non-permeabilized section with MPO in Supplementary Fig. 6E.) (b) Western blot analysis of knee joint homogenates from eight different mice (four joints/group) confirming dramatic decreases in cit-H3 in joints injected with DTA-64 as compared to control aptamer.

Figure 4. DEK is required for NET formation in murine neutrophils.

Neutrophils were purified from the bone marrow of WT (n=3) or Dek-KO (n=3) mice and fixed and stained with DAPI (blue), rabbit anti-DEK (red) and mouse anti-neutrophil elastase (green). (a) Unstimulated neutrophils from BM WT and Dek-KO mice. (b) Neutrophils from WT mice stimulated with LPS for 2 h form NETs, whereas those from Dek-KO mice do not. (c) Addition of recombinant mouse DEK (3.5 μg ml⁻¹) prior to LPS stimulation of Dek-KO neutrophils leads to the formation of NETs (× 40 magnification, scale bar 500 μm). (d) Percentage of neutrophils forming NETs per field in WT neutrophils as compared to neutrophils from Dek-KO mice with and without addition of recombinant DEK, as determined by two-tailed, unequal variance Student's t-test (error bars, s.e.m.). Results shown are representative of at least eight different fields counted for each condition, and represent three independent experiments counted by three different investigators. (e) Extracellular DEK does not enter the nucleus of neutrophils. BM neutrophils obtained from Dek-KO mice were incubated with recombinant DEK (3.5 μg ml⁻¹) prior to PMA stimulation. Cells were fixed, permeabilized and stained with a monoclonal antibody to DEK (red), and the nuclear envelope was stained with an antibody directed against Lamin B (green). (× 60 magnification, scale bar 10 μm). Recombinant DEK is detected only on the cell surface and associated with NETs.

Figure 5. Extracellular recombinant DEK does not enter the cell but restores NET formation in Dek-KO neutrophils.

Dek-KO (n=3) BM neutrophils were treated with recombinant DEK prior to PMA stimulation. (a) Cells were fixed 2-3 h after PMA treatment, and then permeabilized and stained for DEK (red) and wheat germ agglutinin (WGA), a membrane marker. DEK is detected outside of the cell in the NETs (× 60 magnification, scale bar 20 μm, Nikon Confocal). (b) Cells were also stained for the NET marker myeloperoxidase (MPO, purple), DEK (red) and WGA (green). DEK is detected outside of the cell in NETs (× 60 magnification, scale bar 10 μm, Nikon Confocal). Images are representative of three different fields from at least three independent experiments.

Figure 6. DEK is released into the extracellular space by human neutrophils and is found in NETs.

1 × 10⁷ human neutrophils (from two different healthy individuals) in serum-free media were left unstimulated or were stimulated with E. coli (a) or PMA (b). Supernatants and cells were harvested after 2 h of incubation and were analysed by immunoblotting using a rabbit polyclonal antibody specific for DEK. DEK is detected as a 45 kDa and/or a 35 kDa protein, the latter of which is a well-known naturally occurring breakdown product of DEK. The 60 kDa form of DEK is most likely the result of posttranslational modifications (see discussion in text). Results are representative of neutrophils from at least three different healthy human volunteers. (c) Neutrophils were isolated from the peripheral blood of healthy volunteers (upper three panels) or from the synovial fluid of a JIA patient (lower panel). Unstimulated neutrophils, or LPS or PMA-stimulated neutrophils, were stained with Hoechst (blue) for DNA, mouse or rabbit antibody to DEK (red), rabbit anti-neutrophil elastase (green) or mouse anti-LL-37 antibody (magnification × 63, scale bar 10 μm). The results of the experiments shown are representative of those seen with the neutrophils from the synovial fluids of three patients with JIA. (d) DEK autoantibodies purified from the synovial fluid of a JIA patient recognize DEK (red) in spontaneously formed NETs from synovial fluid neutrophils. DNA is stained with DAPI (blue) and the NET-specific marker elastase is detected by antibody and stained green (magnification × 60, scale bar 50 μm). All pictures were taken by confocal microscopy (Nikon Confocal microscope). The lower panel zooms in on the NETs marked by the arrows in the upper panel.

Figure 7. Neutrophils treated with anti-DEK aptamer (DTA-64) have fewer NET structures and show retention of intracellular DEK.

(a) Peripheral blood human neutrophils were obtained from healthy donors and plated on glass coverslips. Control or anti-DEK aptamers (DTA-64) were added to neutrophil cultures 15 min prior to PMA stimulation (10 ng ml⁻¹). Neutrophils were incubated for 1 h at 37 °C, then fixed and stained for NETs with anti-MPO (green), DAPI for DNA (blue) and DEK (red) as shown in representative confocal images from experiments using 5 ng ml⁻¹ of control aptamer or DTA-64 (× 60 magnification, scale bar 20 μm). The inserts zoom in on the NETs marked by the arrows in the upper panel. (b) NET staining intensity based on MPO and DAPI was determined by Metamorph 7.7. Results shown represent the average from the neutrophils of five different healthy donors. The number of NETs/nucleus is markedly reduced (*P=0.00019) in neutrophils treated with anti-DEK aptamer as compared to control aptamer as determined by two-tailed, unequal variance Student's t-test (error bars, s.e.m.).

Figure 8. DEK aptamers block NET formation by activated human neutrophils after 4 h of stimulation with PMA.

(a) Peripheral blood human neutrophils were obtained from healthy individuals and plated on glass coverslips. Aptamers to DEK were added at 50 ng to the neutrophil culture prior to PMA stimulation (10 ng ml⁻¹). Neutrophils were incubated for 4 h at 37 °C, then fixed and stained for NETs with anti-MPO antibodies (green) and DAPI for DNA (blue) (× 20 magnification, scale bar 100 μm). (b) Percentage of neutrophils forming NETs per field following treatment with control library aptamer as compared to neutrophils treated with anti-DEK aptamer (DTA 64) (*P<0.001 as determined by two-tailed, unequal variance Student's t-test ) (error bars, s.e.m.). Results shown are based on three different experiments from three different healthy donors and a total of 13 different fields counted by two different investigators.