An injectable hydrogel to disrupt neutrophil extracellular traps for treating rheumatoid arthritis

Rheumatoid arthritis (RA), an autoimmune disease, is characterized by inflammatory cell infiltration that damages cartilage, disrupts bone, and impairs joint function. The therapeutic efficacy of RA treatments with the severely affected side remains unsatisfactory despite current treatment methods that primarily focus on anti-inflammatory activity, largely because of the complicatedly pathological mechanisms. A recently identified mechanism for RA development involves the interaction of RA autoantibodies with various proinflammatory cytokines to facilitate the formation of neutrophil extracellular traps (NETs), which increased inflammatory responses to express inflammatory cytokines and chemokines. Therefore, NETs architecture digestion may inhibit the positive-feedback inflammatory signal pathway and lessen joint damage in RA. In this work, deoxyribonuclease I (DNase) is connected to oxidized hyaluronic acid (OHA) via Schiff base reaction to extend the half-life of DNase. The modification does not influence the DNase activity for plasmid deoxyribonucleic acid hydrolysis and NETs’ architecture disruption. Carboxymethyl chitosan is crosslinked with DNase-functionalised OHA (DHA) to form an injectable, degradable, and biocompatible hydrogel (DHY) to further strengthen the adhesive capability of DHA. Importantly, the collagen-induced arthritis model demonstrates that intra-articular injection of DHY can significantly reduce inflammatory cytokine expression and alleviate RA symptoms, which can be significantly improved by combining methotrexate. Here, a DNase-functionalised hydrogel has been developed for RA treatment by constantly degrading the novel drug target of NETs to decrease inflammatory response in RA.</p

Opa expression has no impact on the permeability of polarized epithelial monolayer infected with GC.

<p>Polarized T84 cells were apically incubated with GC for 6 h in the presence of 1 μg/ml of FITC or HRP in the apical medium. The fluorescence intensity of FITC in the basolateral media was measured at 490 nm using a fluorimeter. The enzymatic activity of HRP was measured using a color changing substrate. Shown are the average percent of FITC and HRP leaked into the basolateral media from three independent experiments.</p

Opa expression changes gonococcal interaction with polarized epithelial cells.

<p>Polarized T84 cells on transwells were incubated with or without GC MS11Opa+, MS11ΔOpa, and MS11OpaH for 4 h. Cells were fixed, permeabilized, stained for the junctional protein ZO-1 and gonococci, and analyzed using a confocal microscope. Images shown are representative images from three independent experiments. Scale bar, 5 µm.</p

Opa expression reduces the kinetics and magnitude of GC transmigration.

<p>(A-C) Polarized T84 cells grown on transwells were incubated with GC apically at a MOI of 10:1 for varying periods of times. The basolateral media were plated onto GCK to enumerate the number of GC that transmigrated into the basolateral media (A and C). Epithelial cells were lysed and plated to determine the number of adhered GC (B). Shown is the average CFU per transwell (A-B) or the average percentage of transmigrated GC over total epithelial-associated GC (C) from three independent experiments, each of which performed in triplicate. *<i>p</i>< 0.05. (D) The cells were fixed and stained for GC and ZO1. Images of z-series were acquired using a confocal microscope. Shown are xy images at the apical junction areas (top) and xz images from 3D reconstitution across both apical and basolateral surfaces (bottom) from three independent experiments. Scale bar, 10 μm. (E) The cells were processed for transmission electronic microscopy. Arrows, GC. Scale bar, 2 μm.</p

Effects of Opa and pili on GC adherence to and transmigration across polarized epithelial cells.

<p>(A-B) Human endocervical tissue explants were incubated with piliated MS11Opa+, ΔOpa, OpaH, or OpaC for 24 h, stained for ZO-1, nuclei and GC, and analyzed by 3D-CFM (A). GC subepithelial penetration (arrows) was quantified using 3D-CFM images as the percentage of epithelial cells with basal GC staining among the total number of GC-associated epithelial cells (B). Shown are the average values (±SD) of >50 epithelial cells of endocervical tissue explants from two to three human subjects. (C and D) Polarized HEC-1-B cells were apically incubated with piliated MS11Opa+ or ΔOpa. The basal medium was collected after 6 h to determine transmigrated GC (C). The epithelial cells were lysed after 3 h incubation and washing to quantify adherent GC (D). (E-F) Polarized T84 cells were apically incubated with piliated or non-piliated MS11Opa+ or ΔOpa for 6 or 3 h, and the numbers of transmigrated (E) and adherent GC (F) were determined as described above. Shown are the means (±SD) of >6 transwells from 4–6 independent experiments. ***<i>p</i> ≤0.001; **<i>p</i> ≤ 0.01; *<i>p</i>≤0.05.</p

GC induce exfoliation of polarized epithelial cells from human endocervical tissue explants and T84 monolayers while the expression of CEACAM-binding Opa suppresses the exfoliation.

<p>Human endocervical tissue pieces (A-C) and polarized T84 monolayers (A, D, and E) were apically incubated with piliated MS11Opa+, ΔOpa, OpaH, or OpaC at a MOI of ~10 for 6 or 24 h at 37°C, with unassociated GC washed off at 6 and 12 h. Cells were fixed, stained for DNA and GC, and analyzed using 3D-CFM. Shown are representative images that intercept both the apical and basolateral surfaces (Scale bar, 10 μm) (A, B, and D). Based on cell nuclear staining, the average percentage (±SD) of exfoliated epithelial cells was determined by counting the number of epithelial cells localizing above the endocervical epithelium (A-C) and T84 monolayers (A, D, and E), indicated by white dash lines, versus the total number of epithelial cells. Shown are the results from >15 randomly selected fields (>50 cells) from three independent experiments or cervixes of two to three human subjects. ***<i>p</i> ≤0.001; **<i>p</i> ≤ 0.01; *<i>p</i>≤0.05.</p

Inhibition of Ca²⁺ signal and NMII activation reduces GC penetration into the epithelium but not GC adherence and invasion.

<p>(A-C) Polarized T84 cells were untreated or pre-treated with the ROCK inhibitor Y27632 (Y), the MLCK inhibitors ML-7 (M) and PIK, or the intracellular Ca²⁺ release inhibitor 2APB (10 μM), and apically incubated with piliated MS11Opa+ or ΔOpa for 6 h in the presence or absence of inhibitors. The basal medium was collected to determine transmigrated GC (A). Invaded (B) and adhered GC (C) were quantified by the gentamicin resistance assay. (D) Human endocervical tissue explants were incubated with piliated MS11ΔOpa for 24 h in the presence or absence of ML-7 and PIK. GC subepithelial penetration was quantified using 3D-CFM images as the percentage of epithelial cells with basal GC staining among the total number of GC-associated epithelial cells. Shown are the average values (±SD) of >50 epithelial cells of endocervical tissue explants from two to three human subjects. ***<i>p</i> ≤0.001; **<i>p</i> ≤ 0.01; *<i>p</i>≤0.05.</p

GC induce exfoliation of polarized epithelial cells requires the activation of Ca²⁺ flux and Non-muscle Myosin II (NMII) by Myosin Light Chain Kinase (MLCK).

<p>Polarized T84 cells (A and B) and human endocervical tissue explants (C and D) were untreated or pre-treated with the ROCK inhibitor Y27632 (Y, 10 μM) or the MLCK inhibitors ML-7 (M, 10 μM) or PIK (100 μM) for 1 h and apically incubated with piliated MS11Opa+ or ΔOpa for 6 h or 24 h in the presence or absence of inhibitors. Cells were fixed, stained for DNA and GC, and analyzed using 3D-CFM. Shown are representative images (Scale bar, 10 μm) (A and C). The average percentages (±SD) of exfoliated cells were determined as <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006269#ppat.1006269.g001" target="_blank">Fig 1</a> from >15 randomly selected fields (>50 cells) of three independent experiments or cervixes of two to three human subjects. ***<i>p</i> ≤0.001; **<i>p</i> ≤ 0.01; *<i>p</i>≤0.05.</p

GC interaction increases the levels of phosphorylated MLC at the apical junction and apical membrane.

<p>(A-E) Polarized T84 cells were untreated or pre-treated with the ROCK inhibitor Y27632 (Y) and the MLCK inhibitor ML-7 (M) and PIK, and apically incubated with piliated and non-piliated MS11Opa+ and ΔOpa for 6 h in the presence or absence of inhibitors. Cells were stained for phosphorylated MLC (pMLC) and GC and analyzed using 3D-CFM. FIRs of pMLC at the apical to lateral region (A-C) and at the junctional to non-junctional region (D and E) were determined. Shown are representative xz (A) and xy images at the apical junctional location (D), FI maps (D, right panels), and the average FIR (±SD) (B, C, and E) of >50 cells from three independent experiments. Arrows indicate GC. Bar, 5 μm. (F-H) Polarized T84 cells were treated with inhibitors and incubated with piliated GC as above. Cells were then lysed and analyzed by Western blot probing for MLC, pMLC and β-tubulin. The blot was quantified by Phosphorimager. Shown are representative blots (F) and the average fold of increase (±SD) in pMLC:MLC (G) and MLC:tubulin (H) ratios over no GC control from three independent experiments. ***<i>p</i> ≤0.001; **<i>p</i> ≤ 0.01; *<i>p</i>≤0.05.</p

GC induce apical junction disruption in polarized epithelial cells and endocervical tissue explants in a Ca²⁺- and MLCK-dependent manner.

<p>(A and B) Effects of NMII kinase and Ca²⁺ inhibitors on the distribution of E-cadherin. Polarized T84 cells were untreated or pre-treated with NMII kinase inhibitors, Y27632 (Y) and ML-7 (M), or a Ca²⁺ inhibitor, 2APB, and then apically incubated with piliated MS11Opa+ or ΔOpa for 6 h in the presence or absence of inhibitors. (A) Cells were fixed, stained for E-cadherin (E-Cad) and GC, and analyzed using 3D-CFM. (B) The average fluorescence intensity ratios (FIR) (±SD) of E-Cad staining at the cytoplasmic to the cell-cell junctional region was determined from >50 cells of three individual experiments using the NIH ImageJ software. (C) The expression levels of ZO1 in MS11Pil+Opa+- or ΔOpa-infected T84 cells were compared using Western blotting and quantified by the average fold of increases (±SD) in the ratio of ZO1 to tubulin in cell lysates from three independent experiments. (D-F) Effects of MLCK and Ca²⁺ inhibitors on the membrane lateral movement over the apical junction. (D) Polarized T84 cells treated with inhibitors as above were apically inoculated with fluorescently labeled piliated GC for 4 h and basolaterally stained with CellMask for 15 min. (E) Time lapse xz images were acquired using CFM. (F) The average percentage (±SD) of cells showing the basolaterally stained dye moving over to the apical surface was determined from >50 randomly selected cells of three independent experiments. Scale bar, 5 μm. (G-H) Effects of GC and the MLCK inhibitor ML-7 on the ZO1 distribution in human endocervical tissue explants. The tissue explants were untreated or pre-treated with ML-7 (M) for 1 h and incubated with MS11Pil+ΔOpa for 24 h in the absence or presence of ML-7. Tissues were stained for ZO1, DNA, and GC. (G) Shown are representative CFM (left panels, arrows to GC) and 3D reconstituted images (right panels) (Bar, 10 μm). (H) The average percentages (±SD) of GC-associated cells showing continuous ZO1 staining at the apical region were determined from >15 randomly selected fields (>50 cells) from cervixes of two to three human subjects. ***<i>p</i> ≤0.001; **<i>p</i> ≤ 0.01; *<i>p</i>≤0.05. (I) Representative CFM images of ZO1 distribution in exfoliating and surrounding epithelial cells (arrows) of cervical tissues from 4 human subjects.</p