Macrophage-derived reactive oxygen species protects against autoimmune priming with a defined polymeric adjuvant

Abstract

Understanding the nature of adjuvants and the immune priming events in autoimmune diseases, such as rheumatoid arthritis, is a key challenge to identify their aetiology. Adjuvants are, however, complex structures with inflammatory and immune priming properties. Synthetic polymers provide a possibility to separate these functions and allow studies of the priming mechanisms in vivo. A well-balanced polymer, poly-N-isopropyl acrylamide (PNiPAAm) mixed with collagen type II (CII) induced relatively stronger autoimmunity and arthritis compared with more hydrophilic (polyacrylamide) or hydrophobic (poly-N-isopropylacrylamide-co-poly-N-tertbutylacrylamide and poly-N-tertbutylacrylamide) polymers. Clearly, all the synthesized polymers except the more hydrophobic poly-N-tertbutylacrylamide induced arthritis, especially in Ncf1-deficient mice, which are deficient in reactive oxygen species (ROS) production. We identified macrophages as the major infiltrating cells present at PNiPAAm-CII injection sites and demonstrate that ROS produced by the macrophages attenuated the immune response and the development of arthritis. Our results reveal that thermo-responsive polymers with high immune priming capacity could trigger an autoimmune response to CII and the subsequent arthritis development, in particular in the absence of NOX2 derived ROS. Importantly, ROS from macrophages protected against the autoimmune priming, demonstrating a critical regulatory role of macrophages in immune priming events.

Keywords: adjuvant; arthritis; collagen type II; macrophages; poly-N-isopropylacrylamide.

Figure 1

A balance of hydophobicity/hydrophilicity defines the adjuvant property of a polymer. Chemical structure of polymers tested as adjuvants in experimental arthritis are shown (a). Tumour necrosis factor‐α (TNF‐α) analysis at day 21 in serum of mice injected with polymers to assess the systemic biocompatibility of the polymers (b). Arthritis was induced in mice (n = 24 mice per group) using collagen II (CII) and an adjuvant. Mean arthritis score (c) with different polymeric adjuvants mixed with CII in 7‐ to 8‐week‐old B10Q.Ncf1 mice. The maximum score per mouse was 60 points. CII (1 mg/ml) was mixed with each polymer separately and 200 μg of antigen–polymer mixture (1 : 1) was injected intradermally on day 0 and boosted with 100 μg of respective antigen–polymer mixture (1 : 1) on day 21. Only mice that developed arthritis were used for calculations. Anti‐CII antibody levels, before (day 21) and after (day 50) booster immunization of mice (d) were shown. Interferon‐γ (IFN‐γ) (e) concentration in the supernatant of cultured splenocytes re‐stimulated with various stimulants. Error bars indicate ± SEM. *P < 0·05; **P < 0·005 and ***P < 0·0005. n indicates number of mice used in each group.

Figure 2

Polymer–collagen II (CII) binding characteristics and adjuvant potential of different formats of the polymer. To assess the effect of temperature‐dependent phase transition of CII, circular dichroism spectra of poly‐N‐isopropylacrylamide (PNiPAAm) and polyacrylamide (PAAm) with CII mixture (a) at 20° (below the lower critical solution temperature (LCST) of PNiPAAm), and 35° (b, above the LCST of PNiPAAm) were recorded. CII and PNiPAAm spectra were used as controls. Spectra were monitored in the far UV (193–250 nm) region using CII (0·01 mg/ml) and JASCO J600A spectropolarimeter. Polymers (0·01%, weight/volume) in 0·02 m phosphate buffer, pH 7·4 were mixed with CII at 20°C. To analyse the format effect on adjuvant property, two different PNiPAAm formats (linear and microgel) were synthesized and tested in 7‐ to 8‐week‐old B10Q.Ncf1 male mice. Mean arthritis score on different days (c) and anti‐CII responses in the sera collected at days 21 and 50 (d) were shown. Two groups of mice, PNiPAAm‐CII (n = 15) and PNiPAAm microgel‐CII (n = 15), were used. Mice were immunized with 100 μg of CII + 100 μg of PNiPAAm in linear or microgel format and boosted on day 21 with 50 μg of respective antigen–polymer format. Error bars denote ± SEM. *P < 0·05; **P < 0·005 and ***P < 0·0005. Represented results are from two independent experiments and all the animals were used for analysis. n indicates the number of mice used in each group. The maximum score per mouse was 60 points.

Figure 3

Macrophage specific reactive oxygen species (ROS) attenuates poly‐N‐isopropyla‐crylamide–collagen II (PNiPAAm‐CII) ‐induced arthritis. Immunohistochemistry of surrounding tissues at the PNiPAAm‐CII injection site was shown. Immunostaining of macrophage cells at polymer–tissue interface (a). Intracellular staining for cytokines, interferon‐γ (IFN‐γ) (b), interleukin‐4 (IL‐4) (c) and IL‐17 (d), to understand the nature of the infiltrating cells. All the images were taken at 20 × magnification. Adjuvant potential of PNiPAAm in 7‐ to 8‐week‐old B10Q.Ncf1 (ROS production is reduced in all the cells) and MN.B10Q.Ncf1 (ROS production is reduced in all the cells except macrophages) mice was analysed. Mice were divided into four groups complete Freund's adjuvant (CFA) ‐CII (B10Q.Ncf1, n = 10), CFA‐CII (MN.B10Q.Ncf1, n = 10), PNiPAAm‐CII (B10Q.Ncf1, n = 15) and PNiPAAm‐CII (MN.B10Q.Ncf1, n = 15). Mice were immunized with 100 μg of CII + PNiPAAm/CFA and boosted on day 21 with 50 μg of CII + PNiPAAm/incomplete Freund's adjuvant (IFA). Anti‐CII antibody response (e) and mean arthritis score (f) were shown. The maximum score per mouse was 60 points. Error bars denote ± SEM. *P < 0·05; **P < 0·005 and ***P < 0·0005. Represented results are from two independent experiments and all the animals were used for analysis. n indicates the number of mice used in each group.