Adjuvant properties of a biocompatible thermo-responsive polymer of N -isopropylacrylamide in autoimmunity and arthritis

Abstract

To evaluate the thermo-responsive poly(N-isopropylacrylamide) (PNiPAAm) polymer as an adjuvant, we synthesized PNiPAAm through free radical polymerization and characterized it both in vitro and in vivo. The polymer when mixed with collagen type II (CII) induced antigen-specific autoimmunity and arthritis. Mice immunized with PNiPAAm-CII developed significant levels of CII-specific IgG response comprising major IgG subclasses. Antigen-specific cellular recall response was also enhanced in these mice, while negligible level of IFN-g was detected in splenocyte cultures, in vitro. PNiPAAm-CII-immunized arthritic mouse paws showed massive infiltration of immune cells and extensive damage to cartilage and bone. As determined by immunostaining, most of the CII protein retained its native configuration after injecting it with PNiPAAm in naive mice. Physical adsorption of CII and the highmolecular-weight form of moderately hydrophobic PNiPAAm induced a significant anti-CII antibody response. Similar to CII, mice immunized with PNiPAAm and ovalbumin (PNiPAAm-Ova) induced significant anti-ovalbumin antibody response. Comparable levels of serum IFN-g, IL-1b and IL-17 were observed in ovalbumin-immunized mice with complete Freund, incomplete Freund (CFA and IFA) or PNiPAAm adjuvants. However, serum IL-4 levels were significantly higher in PNiPAAm-Ova and CFA-Ova groups compared with the IFA-Ova group. Thus, we show for the first time, biocompatible and biodegradable thermo-responsive PNiPAAm can be used as an adjuvant in several immunological applications as well as in better understanding of the autoimmune responses against self-proteins.

Collagen Type II and a Thermo-Responsive Polymer of N-Isopropylacrylamide Induce Arthritis Independent of Toll-Like Receptors

“…Recently we evaluated the adjuvant property of PNiPAAm having Mw 120 kDa in mice using two different antigens. 25 When the CII was mixed with PNiPAAm, most of the CII protein retained its native configuration. We tested the adjuvant efficiency of PNiPAAm with molecular weights of 70 and 120 kDa, and found that physical adsorption and high molecular weight (120 kDa) form of moderately hydrophobic PNiPAAm seems to be more optimal to induce a significant adaptive immune response.…”

“…We tested the adjuvant efficiency of PNiPAAm with molecular weights of 70 and 120 kDa, and found that physical adsorption and high molecular weight (120 kDa) form of moderately hydrophobic PNiPAAm seems to be more optimal to induce a significant adaptive immune response. 25 Although PNiPAAm does not have any known pathogen-associated molecular pattern (PAMP) ligands, it still induces a broad range of cytokine response (IL-1␤, IFN-␥, IL-4, and IL-17). Thus, PNiPAAm offers opportunities to study the actual autoimmune responses to self-protein(s).…”

“…Antigen-specific immune responses were observed when the synthetic polymer of N-isopropylacrylamide was used as an adjuvant. 25 To understand the actual immune responses to an autoantigen, we immunized mice with CII mixed with PNiPAAm devoid of any classical adjuvant. We measured serum IFN-␥, IL-4, and IL-17 levels as an indicator of activation of three major T-helper cell populations (Th1, Th2, and Th17).…”

“…However, these adjuvants often tend to strongly change the ensuing immune response, thereby precluding our understanding of the ac- tual immune response to the self-protein. Because we observed the adjuvant property of the synthetic PNiPAAm in B10.RIII mice, 25 we tested a series of inbred mouse strains for arthritis susceptibility with the polymeric adjuvant and CII to re-evaluate the MHC restriction of CIIinduced arthritis. Many of the strains developed highly frequent and severe arthritis ( Figure 3A and B) with a pattern similar to classical CIA 3,7,43 ; that is, mice with the MHC haplotype (H-2 q ) were highly susceptible (cumulative incidence of arthritis in H-2 q versus other MHC haplotypes, P Ͻ 0.0001), with a more severe arthritis phenotype (mean maximal score, P Ͻ 0.0001) and a robust adaptive immune response ( Figure 3C), whereas mice with other MHC haplotypes (H-2 p, b, d ) were resistant, with low anti-CII antibody levels (d35, P Ͻ 0.05 and d70, P Ͻ 0.0001).…”

“…Recently, we identified a synthetic thermo-responsive polymer of N-isopropylacrylamide (PNiPAAm), which is biocompatible and modifiable to study autoimmunity and autoimmune diseases. 25 PNiPAAm has a lower critical solution temperature (LCST) of precipitation, around 32.5°C in water, and changes reversibly from hydrophilic to hydrophobic around this temperature. 26 This reversible phase-transition property of PNiPAAm was used for the colloid suspension formation together with CII to induce arthritis in mice.…”