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

2011; Elsevier BV; Volume: 179; Issue: 5 Linguagem: Inglês

10.1016/j.ajpath.2011.07.034

ISSN

1525-2191

Autores

Akhilesh Kumar Shakya, Ashok Kumar, Dorota Klaczkowska, Malin Hultqvist, Kristin Hagenow, Rikard Holmdahl, Kutty Selva Nandakumar,

Tópico(s)

Proteoglycans and glycosaminoglycans research

Resumo

We established and characterized an arthritis mouse model using collagen type II (CII) and a thermo-responsive polymer, poly(N-isopropylacrylamide) (PNiPAAm). The new PNiPAAm adjuvant is TLR-independent, as all immunized TLR including MyD88-deficient mice developed an anti-CII response. Unlike other adjuvants, PNiPPAm did not skew the cytokine response (IL-1β, IFN-γ, IL-4, and IL-17), as there was no immune deviation towards any one type of immune spectrum after immunization with CII/PNiPPAm. Hence, using PNiPAAm, we studied the actual immune response to the self-protein, CII. We observed arthritis and autoimmunity development in several murine strains having different major histocompatibility complex (MHC) haplotypes after CII/PNiPAAm immunization but with a clear MHC association pattern. Interestingly, C57Bl/6 mice did not develop CII-induced arthritis, with PNiPAAm demonstrating absolute requirement for a classical adjuvant. Presence of a gene (Ncf1) mutation in the NADPH oxidation complex has a profound influence in arthritis and using PNiPAAm we could show that the high CIA severity in Ncf1 mutated mice is independent of any classical adjuvant. Macrophages, neutrophils, eosinophils, and osteoclasts but not mast cells dominated the inflamed joints. Furthermore, arthritis induction in the adjuvant-free, eosinophil-dependent Vβ12 DBA/1 mice could be shown to develop arthritis independent of eosinophils using CII/PNiPAAm. Thus, biocompatible and biodegradable PNiPAAm offers unique opportunities to study actual autoimmunity independent of TLR and a particular cytokine phenotype profile. We established and characterized an arthritis mouse model using collagen type II (CII) and a thermo-responsive polymer, poly(N-isopropylacrylamide) (PNiPAAm). The new PNiPAAm adjuvant is TLR-independent, as all immunized TLR including MyD88-deficient mice developed an anti-CII response. Unlike other adjuvants, PNiPPAm did not skew the cytokine response (IL-1β, IFN-γ, IL-4, and IL-17), as there was no immune deviation towards any one type of immune spectrum after immunization with CII/PNiPPAm. Hence, using PNiPAAm, we studied the actual immune response to the self-protein, CII. We observed arthritis and autoimmunity development in several murine strains having different major histocompatibility complex (MHC) haplotypes after CII/PNiPAAm immunization but with a clear MHC association pattern. Interestingly, C57Bl/6 mice did not develop CII-induced arthritis, with PNiPAAm demonstrating absolute requirement for a classical adjuvant. Presence of a gene (Ncf1) mutation in the NADPH oxidation complex has a profound influence in arthritis and using PNiPAAm we could show that the high CIA severity in Ncf1 mutated mice is independent of any classical adjuvant. Macrophages, neutrophils, eosinophils, and osteoclasts but not mast cells dominated the inflamed joints. Furthermore, arthritis induction in the adjuvant-free, eosinophil-dependent Vβ12 DBA/1 mice could be shown to develop arthritis independent of eosinophils using CII/PNiPAAm. Thus, biocompatible and biodegradable PNiPAAm offers unique opportunities to study actual autoimmunity independent of TLR and a particular cytokine phenotype profile. Rheumatoid arthritis (RA) is a debilitating multifactorial disease involving complex genetic and immunological interactions. Immunization with collagen type II (CII) in an adjuvant induces polyarthritis in susceptible strains of rodents and primates, known as the collagen-induced arthritis (CIA) model, which is widely used for studying arthritis development. CIA resembles RA in several aspects, and is characterized by synovial hyperplasia, infiltration of immune cells, marginal erosions, and cartilage destruction with disruption of joint and cartilage architecture. As in RA, susceptibility to CIA is associated with certain major histocompatibility complex (MHC) class II alleles,1Brunsberg U. Gustafsson K. Jansson L. Michaëlsson E. &Ährlund-Richter L. Pettersson S. Mattsson R. Holmdahl R. Expression of a transgenic class II Ab gene confers susceptibility to collagen-induced arthritis.Eur J Immunol. 1994; 24: 1698-1702Crossref PubMed Scopus (130) Google Scholar, 2Rosloniec E.F. Brand D.D. Myers L.K. Whittington K.B. Gumanovskaya M. Zaller D.M. Woods A. Altmann D.M. Stuart J.M. Kang A.H. An HLA-DR1 transgene confers susceptibility to collagen-induced arthritis elicited with human type II collagen.J Exp Med. 1997; 185: 1113-1122Crossref PubMed Scopus (198) Google Scholar H2q and H2r haplotypes being the most arthritis-permissive.3Wooley P.H. Luthra H.S. Stuart J.M. David C.S. Type II collagen-induced arthritis in mice I. Major histocompatibility complex (I region) linkage and antibody correlates.J Exp Med. 1981; 154: 688-700Crossref PubMed Scopus (592) Google Scholar, 4Wooley P.H. Luthra H.S. Griffiths M.M. Stuart J.M. Huse A. David C.S. Type II collagen induced arthritis in mice IV. Variations in immunogenetic regulation provide evidence for multiple arthritogenic epitopes on the collagen molecule.J Immunol. 1985; 135: 2443-2451PubMed Google Scholar However, C57Bl/6 mice with H2b haplotype also develop arthritis,5Campbell I.K. Hamilton J.A. Wicks I.P. Collagen-induced arthritis in C57BL/6 (H-2b) mice: new insights into an important disease model of rheumatoid arthritis.Eur J Immunol. 2000; 30: 1568-1575Crossref PubMed Scopus (179) Google Scholar but influence of the adjuvant on arthritis development in these mice has not been clarified. The gene underlying the susceptibility within the H2q haplotype is Aq1Brunsberg U. Gustafsson K. Jansson L. Michaëlsson E. &Ährlund-Richter L. Pettersson S. Mattsson R. Holmdahl R. Expression of a transgenic class II Ab gene confers susceptibility to collagen-induced arthritis.Eur J Immunol. 1994; 24: 1698-1702Crossref PubMed Scopus (130) Google Scholar and the responsible MHC bound peptide is the CII260-270 peptide.6Michaëlsson E. Andersson M. Engstrm̈ A. Holmdahl R. Identification of an immunodominant type-II collagen peptide recognized by T cells in H-2q mice: self tolerance at the level of determinant selection.Eur J Immunol. 1992; 22: 1819-1825Crossref PubMed Scopus (119) Google Scholar The peptide is conserved in CII, and consequently, severe arthritis is induced after immunization with various heterologous (eg, chick, human, bovine, or rat) CII.7Holmdahl R. Jansson L. Andersson M. Larsson E. Immunogenetics of type II collagen autoimmunity and susceptibility to collagen arthritis.Immunology. 1988; 65: 305-310PubMed Google Scholar Murine CII differs by one amino acid, affecting MHC binding,6Michaëlsson E. Andersson M. Engstrm̈ A. Holmdahl R. Identification of an immunodominant type-II collagen peptide recognized by T cells in H-2q mice: self tolerance at the level of determinant selection.Eur J Immunol. 1992; 22: 1819-1825Crossref PubMed Scopus (119) Google Scholar and induction of mouse CII requires stronger adjuvant and more susceptible genetic backgrounds.8Courtenay J.S. Dallman M.J. Dayan A.D. Martin A. Mosedal B. Immunization against heterologous type II collagen induces arthritis in mice.Nature. 1980; 283: 666-667Crossref PubMed Scopus (884) Google Scholar The most commonly used adjuvant (complete Freund's adjuvant; CFA) to induce CIA contains bacterial derivatives, which strongly deviate the ensuing immune response.9Reed S.G. Bertholet S. Coler R.N. Friede M. New horizons in adjuvants for vaccine development.Trends Immunol. 2009; 30: 23-32Abstract Full Text Full Text PDF PubMed Scopus (531) Google Scholar, 10Wilson-Welder J.H. Torres M.P. Kipper M.J. Mallapragada S.K. Wannemuehler M.J. Narasimhan B. Vaccine adjuvants: current challenges and future approaches.J Pharm Sci. 2009; 98: 1278-1316Crossref PubMed Scopus (192) Google Scholar The use of an adjuvant such as CFA in arthritis induction is presumably to break the immune tolerance to the self-protein. The mycobacterial components, having pathogen-associated molecular patterns in CFA, activate antigen-presenting cells via pattern recognition receptors that direct T cells toward a Th1- or Th17- type immune responses characterized by the production of pro-inflammatory cytokines like IFN-γ, and IL-17.11Yip H.C. Karulin A.Y. Tary-Lehmann M. Hesse M.D. Radeke H. Heeger P.S. Trezza R.P. Heinzel F.P. Forsthuber T. Lehmann P.V. Adjuvant-guided type-1 and type-2 immunity: infectious/noninfectious dichotomy defines the class of response.J Immunol. 1999; 162: 3942-3949PubMed Google Scholar, 12Quesniaux V. Fremond C. Jacobs M. Parida S. Nicolle D. Yeremeev V. Bihl F. Erard F. Botha T. Drennan M. Soler M.N. Le Bert M. Schnyder B. Ryffel B. Toll-like receptor pathways in the immune responses to mycobacteria.Microbes Infect. 2004; 6: 946-959Crossref PubMed Scopus (226) Google Scholar On the other hand, incomplete Freund's adjuvant (IFA), without any mycobacterium, deviates the immune response toward Th2 type.13Mauri C. Williams R.O. Walmsley M. Feldmann M. Relationship between Th1/Th2 cytokine patterns and the arthritogenic response in collagen-induced arthritis.Eur J Immunol. 1996; 26: 1511-1518Crossref PubMed Scopus (286) Google Scholar, 14Mauri C. Gray D. Mushtaq N. Londei M. Prevention of arthritis by interleukin 10-producing B cells.J Exp Med. 2003; 197: 489-501Crossref PubMed Scopus (703) Google Scholar However, use of strong adjuvants precludes our understanding of the actual immune responses to the self-protein, CII and associated pathological pathways. It is well documented that several polymeric systems act as adjuvant especially, poly(glycolide) (PGA),15Nellore R.V. Pande P.G. Young D. Bhagat H.R. Evaluation of biodegradable microspheres as vaccine adjuvant for hepatitis B surface antigen.J Parenter Sci Technol. 1992; 46: 176-180PubMed Google Scholar poly(lactide-co-glycolide) (PLGA),16Eldridge J.H. Staas J.K. Meulbroek J.A. Tice T.R. Gilley R.M. Biodegradable and biocompatible poly(DL-lactide-co-glycolide) microspheres as an adjuvant for staphylococcal enterotoxin B toxoid which enhances the level of toxin-neutralizing antibodies.Infect Immun. 1991; 59: 2978-2986PubMed Google Scholar, 17Mutsumi Y. Jessica M. Julia E.B. Effect of poly(lactic-co-glycolic acid) contact on maturation of murine bone marrow-derived dendritic cells.J Biomed Mater Res A. 2007; 80: 7-12PubMed Google Scholar polyacrylic acid (PAA) and its alkyl esters,18Hilgers L.A. Nicolas I. Lejeune G. Dewil E. Strebelle M. Boon B. Alkyl-esters of polyacrylic acid as vaccine adjuvants.Vaccine. 1998; 16: 1575-1581Crossref PubMed Scopus (28) Google Scholar poloxamers,19Hartikka J. Geall A. Bozoukova V. Kurniadi D. Rusalov D. Enas J. Yi J.H. Nanci A. Rolland A. Physical characterization and in vivo evaluation of poloxamer-based DNA vaccine formulations.J Gene Med. 2008; 10: 770-782Crossref PubMed Scopus (28) Google Scholar polyphosphazenes,20Mutwiri G. Benjamin P. Soita H. Townsend H. Yost R. Roberts B. Andrianov A.K. Babiuk L.A. Poly[di(sodium carboxylatoethylphenoxy)phosphazene] (PCEP) is a potent enhancer of mixed Th1/Th2 immune responses in mice immunized with influenza virus antigens.Vaccine. 2007; 25: 1204-1213Crossref PubMed Scopus (97) Google Scholar polyoxyethylene (POE),21Kleine B. Rapp W. Wiesmuller K.H. Edinger M. Beck W. Metzger J. Ataulakhanov R. Jung G. Bessler W.G. Lipopeptide-polyoxyethylene conjugates as mitogens and adjuvants.Immunobiology. 1994; 190: 53-66Crossref PubMed Scopus (21) Google Scholar polyoxypropylene (POP), polyacrylate22Zaman M. Skwarczynski M. Malcolm J.M. Urbani C.N. Jia Z. Batzloff M.R. Good M.F. Monteiro M.J. Toth I. Self-adjuvanting polyacrylic nanoparticulate delivery system for group A streptococcus (GAS) vaccine.Nanomedicine. 2011; 7: 168-173Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar, and chitosan polymers.23Boonyo W. Junginger H.E. Waranuch N. Polnok A. Pitaksuteepong T. Chitosan and trimethyl chitosan chloride (TMC) as adjuvants for inducing immune responses to ovalbumin in mice following nasal administration.J Control Release. 2007; 121: 168-175Crossref PubMed Scopus (83) Google Scholar These biocompatible and biodegradable polymers have advantages over conventional adjuvants, mainly in the manipulation of degradation kinetics, by varying the concentration of monomers and cross-linkers,24Eldridge J.H. Staas J.K. Meulbroek J.A. McGhee J.R. Tice T.R. Gilley R.M. Biodegradable microspheres as a vaccine delivery system.Mol Immunol. 1991; 28: 287-294Crossref PubMed Scopus (381) Google Scholar thereby modulating the physicochemical properties of polymers and leading to different release profiles of an antigen. Recently, we identified a synthetic thermo-responsive polymer of N-isopropylacrylamide (PNiPAAm), which is biocompatible and modifiable to study autoimmunity and autoimmune diseases.25Shakya A.K. Kumar A. Nandakumar K.S. Adjuvant properties of a biocompatible thermo-responsive polymer of N-isopropylacrylamide in autoimmunity and arthritis.J R Soc Interface. 2011; https://doi.org/10.1098/rsif.2011.0114Crossref PubMed Scopus (21) 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.26Shin B.C. Jhon M.S. Lee H.B. Yuk S.H. Temperature-induced phase transition of semi interpenetrating polymer networks composed of poly(N-isopropyl acrylamide) and hydrophilic polymers.Eur Polym J. 1998; 34: 171-174Crossref Scopus (44) Google Scholar This reversible phase-transition property of PNiPAAm was used for the colloid suspension formation together with CII to induce arthritis in mice. Furthermore, our recent studies demonstrated Ncf1 (coding p47phox subunit of the NADPH oxidase complex, which is a multicomponent electron carrier that is responsible for the reduction of oxygen, resulting in the production of ROS) polymorphism as one of the major genetic factors that control arthritis severity and chronicity, regulating autoimmune reactions and impaired tolerance to CII.27Olofsson P. Holmberg J. Tordsson J. Lu S. Akerstrom B. Holmdahl R. Positional identification of Ncf1 as a gene that regulates arthritis severity in rats.Nat Genet. 2003; 33: 25-32Crossref PubMed Scopus (300) Google Scholar, 28Hultqvist M. Olofsson P. Holmberg J. Bäckstrm̈ B.T. Tordsson J. Holmdahl R. Enhanced autoimmunity, arthritis, and encephalomyelitis in mice with a reduced oxidative burst due to a mutation in the Ncf1 gene.Proc Natl Acad Sci USA. 2004; 101: 12646-12651Crossref PubMed Scopus (275) Google Scholar, 29Hultqvist M. Backlund J. Bauer K. Gelderman K.A. Holmdahl R. Lack of reactive oxygen species breaks T cell tolerance to collagen type II and allows development of arthritis in mice.J Immunol. 2007; 179: 1431-1437PubMed Google Scholar In addition, we have also observed a high frequency of arthritis after CII immunization without any adjuvant in a transgenic mice expressing a CII-specific TCR Vβ12 chain that recognizes the immunodominant glycosylated CII260−270 peptide that is dependent on eosinophilic inflammation.30Bockermann R. Holmdahl R. Type II collagen without adjuvant induces eosinophilic arthritis.Eur J Immunol. 2007; 37: 540-548Crossref PubMed Scopus (9) Google Scholar Hence, in the present study, we tested various mouse strains using PNiPAAm-CII immunization to find genetic restriction of arthritis development in the absence of a classical adjuvant. Also, we analyzed adjuvant dependency of strong arthritis promoted by a mutation in Ncf1 and determined the requirement for eosinophilic inflammation for arthritis induction in Vβ12 transgenic mice. Furthermore, we studied the nature of cytokine response induced with PNiPAAm and the self-antigen, CII and compared it with immunization of CII emulsified in Freund's adjuvant(s). Founders of B10.Q/Rhd mice were provided by Professor Jan Klein (Tübingen University, Tübingen, Germany). Breeding pairs of BALB/cJ, C57BL/6J, DBA/1J, C57BL/10ScNJ mice (Tlr4lps-del) and TLR7 knock-out mice were from Jackson Laboratories (Bar Harbor, ME). Tlr4lps-del mice were crossed together with B10.Q/Rhd mice to generate mice expressing arthritis-permissive MHC Aq. B10.Q mice with a mutated Ncf1 gene (B10.Q.Ncf1*/*) have been described previously.28Hultqvist M. Olofsson P. Holmberg J. Bäckstrm̈ B.T. Tordsson J. Holmdahl R. Enhanced autoimmunity, arthritis, and encephalomyelitis in mice with a reduced oxidative burst due to a mutation in the Ncf1 gene.Proc Natl Acad Sci USA. 2004; 101: 12646-12651Crossref PubMed Scopus (275) Google Scholar The Vβ12 transgenic mouse was generated on an SWR background31Mori L. Loetscher H. Kakimoto K. Bluethmann H. Steinmetz M. Expression of a transgenic T cell receptor β chain enhances collagen-induced arthritis.J Exp Med. 1992; 176: 381-388Crossref PubMed Scopus (45) Google Scholar and has been backcrossed to DBA/1J for more than 15N generations. B10.P/Rhd mice were bred at the animal facility of the division of Medical Inflammation Research at Lund University. The C3H.Q/Rhd strain was established in our laboratory by inserting the H-2q haplotype from an older C3H.Q strain into mice of the C3H.NB background.32Kjellen P. Jansson L. Vestberg M. Andersson A.A. Mattsson R. Holmdahl R. The H2-Ab gene influences the severity of experimental allergic encephalomyelitis induced by proteolipoprotein peptide 103–116.J Neuroimmunol. 2001; 120: 25-33Abstract Full Text Full Text PDF PubMed Scopus (8) Google Scholar Rhd indicates that these classic inbred mouse strains have been maintained in our laboratory for more than 2 decades. F1 Intercross mice QD = (B10.Q × DBA/1) F1 and QB = (BALB/c × B10.Q) F1 were bred in MIR, Lund University and used in this study. TLR 1, 2, 3, 5, 6, or 9 and MyD88 knock-out mice backcrossed to C57BL/6 for seven to 10 generations were obtained from Institute for Microbiology, Tumor and Cell Biology (MTC) animal facility, Karolinska Institute, Stockholm, Sweden. Most of the TLR-deficient mice were originally derived by the laboratory of Professor Shizuo Akira, Osaka University, Japan. Age-matched 8- to 12-week-old mice of both sex were used for the experiments. All animals were kept in a climate controlled environment having 12-hour light/dark cycles in polystyrene cages containing wood shavings under specific pathogen-free conditions and were fed standard rodent chow and water ad libitum. Animal welfare authorities (Lund-Malmö and Stockholm regions, Sweden) approved the animal experiments (permit numbers N310-07, M107-07, and N66-10). N-isopropylacrylamide (NiPAAm) was purchased from Acros Organics (Schwerte, Germany). Ammonium persulphate (APS) and N,N,N′,N′-tetramethylethylenediamine (TEMED) were purchased from Sisco Research Laboratories (Mumbai, India). PNiPAAm was synthesized through free radical polymerization by using APS and TEMED. For synthesis of PNiPAAm (1%, w/v), 100 mg of NiPAAm was dissolved in 10 mL of degassed water. Initially, the solution was bubbled with N2 gas for 20 minutes, and free radical polymerization was initiated by adding 15 mg of APS and 19.5 μL of TEMED. The reaction vial was immediately filled with nitrogen and tightly sealed. The reaction was stopped after 18 hours by salt induced thermal precipitation. The thermo-precipitation (40°C) and redissolution (4°C) was done several times to ensure complete removal of unreacted monomers and the polymer was freeze-dried for further use. The molecular weight of synthesized PNiPAAm was determined by gel permeation chromatography (Waters, Milford, MA) using tetrahydrofuran (THF) as an eluent and polystyrene as standard. CII used for immunization was from rat SWARM chondrosarcoma prepared by pepsin digestion and further purified as described earlier.33Grab B. Miles A.J. Furcht L.T. Fields G.B. Promotion of fibroblast adhesion by triple-helical peptide models of type I collagen-derived sequences.J Biol Chem. 1996; 271: 12234-12240Crossref PubMed Scopus (83) Google Scholar Several different mouse strains were tested for arthritis development after PNiPAAm-CII immunization. QD mice were immunized with PNiPAAm-Ova or PNiPAAm alone but they did not show any clinical symptoms for arthritis. CII or ovalbumin protein grade II (Sigma-Aldrich, St. Louis, MO) emulsified in complete Freund adjuvant (CFA; Difco, Detroit, MI) constituted positive and negative controls, respectively. Both CII and ovalbumin proteins (1 mg/mL) were mixed with PNiPAAm separately and 100 μg of antigen–polymer mixture (1:1) was injected s.c. on day 0 and boosted with 50 μg of respective antigen-polymer mixture (1:1) on day 35. Serum samples were collected on different days through the retro-orbital plexus. The amounts of total anti-CII IgG were determined through quantitative ELISA as described earlier.34Holmdahl R. Rubin K. Klareskog L. Larsson E. Wigzell H. Characterization of the antibody response in mice with type II collagen-induced arthritis, using monoclonal anti-type II collagen antibodies.Arthritis Rheum. 1986; 29: 400-410Crossref PubMed Scopus (328) Google Scholar Affinity-purified anti-CII antibody from pooled sera of CII-immunized mice or pooled sera was used as the standard. Biotinylated rat anti-mouse IgG κ (prepared in-house, clone 187.1) or mouse anti-mouse IgG2c (BD), or peroxidase-conjugated goat anti-mouse antibodies specific for IgG1, IgG2b, or IgG3 (Southern Biotech, Birmingham, AL) were used as detecting antibodies. Binding of biotinylated antibodies was revealed by extravidin peroxidase (Sigma-Aldrich). It is noteworthy that B10 mice express an IgG2a-related allele, IgG2c.35Morgado M.G. Cam P. Gris-Liebe C. Cazenave P.A. Jouvin-Marche E. Further evidence that BALB/c and C57BL/6 gamma 2a genes originate from two distinct isotypes.EMBO J. 1989; 8: 3245-3251PubMed Google Scholar Plates were developed using ABTS (Roche Diagnostic Systems) as substrate, and measured at 405 nm (Synergy-2, BioTek Instruments, Winooski, VT). Isotype levels were measured as arbitrary units using pooled sera from arthritic mice as the standard. Mice were examined for the arthritis development twice per week. Scoring of animals was done blindly using a scoring system based on the number of inflamed joints in each paw, inflammation being defined by swelling and redness.36Holmdahl R. Carlsen S. Mikulowska A. Vestberg M. Brunsberg U. Hansson A.-S. Sundvall M. Jansson L. Pettersson U. Genetic analysis of murine models for rheumatoid arthritis.in: Adolpho K.W. CRC press, New York1998: 215-238Google Scholar In this scoring system each inflamed toe or knuckle gives one point, whereas an inflamed wrist or ankle gives five points, resulting in a score of 0 to 15 (five toes + five knuckles + one wrist/ankle) for each paw and 0 to 60 points for each mouse. For immunohistochemistry, mouse paws after decalcification for 3 to 4 weeks in an ethylenediaminetetraacetic acid (EDTA) solution containing polyvinylpyrrolidone (PVP) and Tris (pH 6.9) were frozen in OCT compound using isopentane on dry ice. The samples were stored at −80°C until cryosectioned at 10 μm at −30°C. Sections were stained with safranin to visualize joint and cartilage destruction and mast cells. Osteoclasts were detected using histochemical staining for tartarate-resistant acid phosphatase activity.37Nandakumar K.S. Holmdahl R. Arthritis induced with cartilage-specific antibodiesis IL-4-dependent.Eur J Immunol. 2006; 36: 1608-1618Crossref PubMed Scopus (31) Google Scholar Sections were counterstained with aniline blue (0.1%, w/v). For detection of macrophages and neutrophils, anti-mouse CD11b (M1/70) and rat anti-mouse Ly6G (RB6-8C5) antibodies were used as primary reagents.38Nandakumar K.S. Svensson L. Holmdahl R. Collagen type II specific monoclonal antibody induced arthritis (CAIA) in mice Description of the disease and the influence of age, sex, and genes.Am J Pathol. 2003; 163: 1827-1837Abstract Full Text Full Text PDF PubMed Scopus (221) Google Scholar Either streptavidin or goat anti-mouse peroxidase was used for detection. Diaminobenzidine staining was performed according to established procedures. Eosinophils were identified by staining cyanide-resistant eosinophil peroxidase activity.30Bockermann R. Holmdahl R. Type II collagen without adjuvant induces eosinophilic arthritis.Eur J Immunol. 2007; 37: 540-548Crossref PubMed Scopus (9) Google Scholar Mice immunized with PNiPAAm-CII were used to measure serum IL-1β, IL-4, IFN-γ, and IL-17 levels. IFN-γ levels were determined by coating microtiter plates with anti–IFN-γ (R46-A2) in PBS overnight. After blocking with bovine serum albumin (1%, w/v) in PBS, supernatant was added to these plates. Biotinylated anti–IFN-γ-bio (AN18) was used as secondary antibody. Serum IL-4 (using 11B11 and BVD6-24G2-bio; BD Biosciences), IL-17 (using TC11-18H10 and TCH11-8H4.1-bio, BD Biosciences) and TNF-α (using purified rat anti-mouse TNF-α and biotinylated rat anti-mouse TNF-α; BD Biosciences) levels were measured by ELISA similar to IFN-γ. Recombinant cytokines were used as standards. IL-1β levels were measured using the assay kit supplied by eBioscience. IL-5 was neutralized in vivo by intraperitoneal injection of protein G purified rat monoclonal antibody, TRFK-5 as follows: days −2, 5, and 18 before/after CII immunization with 50 μg of TRFK-5 mixed with 50 μg of affinity purified normal rat serum IgG (control antibody). The control mice received the equivalent amount of control antibody alone. For calculation of arthritis susceptibility and severity, all of the mice were used. The severity of arthritis was analyzed by Mann–Whitney U-test and the incidence by Chi Square test using the Statview 5.0.1 version (SAS Institute, NC). Significance was considered when P < 0.05, for a 95% confidence interval. Poly(N-isopropylacrylamide) was synthesized through free radical polymerization, having weight-average molecular weight of 120 kDa. Antigen-specific immune responses were observed when the synthetic polymer of N-isopropylacrylamide was used as an adjuvant.25Shakya A.K. Kumar A. Nandakumar K.S. Adjuvant properties of a biocompatible thermo-responsive polymer of N-isopropylacrylamide in autoimmunity and arthritis.J R Soc Interface. 2011; https://doi.org/10.1098/rsif.2011.0114Crossref PubMed Scopus (21) 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-γ, IL-4, and IL-17 levels as an indicator of activation of three major T-helper cell populations (Th1, Th2, and Th17). Interestingly, all these three cytokines were found to be elevated in PNiPAAm adjuvant group (Figure 1 A–C). Similarly, we measured serum IL-1β level as a read out for the possible involvement of inflammasome pathway when PNiPAAm was used as an adjuvant and found enhanced IL-1β production in mice after PNiPAAm-CII immunization similar to Freund's adjuvant groups (Figure 1D). However, TNF-α levels in all of the groups during this early phase of CIA (days 10 and 19) were undetectable, suggesting the possibility of different induction kinetics for TNF-α production in these mice. Notably, significant TNF-α expression was reported in CIA mice during or after arthritis onset39Feldmann M. Brennan F.M. Maini R.N. Role of cytokines in rheumatoid arthritis.Annu Rev Immunol. 1996; 14: 397-440Crossref PubMed Scopus (2299) Google Scholar, 40Wei X.Q. Leung B.P. Arthur H.M. McInnes I.B. Liew F.Y. Reduced incidence and severity of collagen-induced arthritis in mice lacking IL-18.J Immunol. 2001; 166: 517-521PubMed Google Scholar but very low levels of TNF-α were detected in CIA mouse joints between days 4 and 1541Rioja I. Bush K.A. Buckton J.B. Dickson M.C. Life P.F. Joint cytokine quantification in two rodent arthritis models: kinetics of expression, correlation of mRNA and protein levels and response to prednisolone treatment.Clin Exp Immunol. 2004; 137: 65-73Crossref PubMed Scopus (100) Google Scholar and in QB mice, the mean day of disease onset was day 57 ± 2.7.42Nandakumar K.S. Lindqvist A.K. Holmdahl R. A dominant suppressive MHC class II haplotype interacting with autosomal genes controls autoantibody production and chronicity of arthritis.Ann Rheum Dis. 2011; 70: 1664-1670Crossref PubMed Scopus (8) Google Scholar To understand further whether PNiPAAm adjuvant acts via the TLR pathway, we first tested TLR4del mice introgressed with H-2q haplotype. TLR4del mice developed significant arthritis compared to that in WT littermate controls (Figure 2A). Despite the arthritis development, the anti-CII antibody response was low (Figure 2B), which might explain the delayed arthritis onset in TLR4del mice. However, severe arthritis in TLR4del mice raised the question as to whether the PNiPAAm acts as a ligand for any other toll-like receptors besides TLR4. Because all of the TLR knock-out mice are in C57Bl/6 background, we decided to check anti-CII antibody response in these mice after PNiPAAm-CII immunization. As shown in Figure 2C, all of the TLR knock-out mice including MyD88-deficient mice developed an anti-CII response. To induce arthritis in the widely used animal model (CIA) for rheumatoid arthritis, mice are injected with either homologous or heterologous CII emulsified in complete or incomplete Freund's adjuvant. However, these adjuvants often tend to strongly change the ensuing immune response, thereby precluding our understanding of the actual immune response to the self-protein. Because we observed the adjuvant property of the synthetic PNiPAAm in B10.RIII mice,25Shakya A.K. Kumar A. Nandakumar K.S. Adjuvant properties of a biocompatible thermo-responsive polymer of N-isopropylacrylamide in autoimmunity and arthritis.J R Soc Interface. 2011; https://doi.org/10.1098/rsif.2011.0114Crossref PubMed Scopus (21) we tested a series of inbred mouse strains for arthritis susceptibility with the polymeric adjuvant and CII to re-evaluate the MHC restriction of CII-induced arthriti

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