Synthetic Polymer as an Adjuvant in Collagen‐Induced Arthritis

Akhilesh Kumar Shakya1, Kutty Selva Nandakumar2

1 Department of Chemical Engineering, Texas Tech University, Lubbock, Texas, 2 University of Arkansas for Medical Sciences, Little Rock, Arkansas
Publication Name:  Current Protocols in Mouse Biology
Unit Number:   
DOI:  10.1002/9780470942390.mo130226
Online Posting Date:  March, 2014
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Abstract

Collagen‐induced arthritis (CIA), the classical animal model for experimental arthritis, resembles human rheumatoid arthritis in several aspects. However, the most widely used method of inducing CIA utilizes Freund's adjuvants, which can skew the elicited immune responses and also pose toxicity problems. This unit describes a new method of inducing CIA using a well defined stimuli‐responsive synthetic polymer, poly‐N‐isopropylacrylamide‐based adjuvant, mixed with the joint cartilage protein collagen type II (CII). PNiPAAm as an adjuvant is biodegradable and biocompatible, and does not skew immune responses. Thus, it is helpful in the development of arthritis models for studying antigen and tissue ‐specific autoimmune responses in an unbiased manner. This model is valuable for analyzing disease pathways, positional identification of genes regulating arthritis, validation of existing therapies, and exploring new therapeutic targets. Furthermore, this newly developed PNiPAAm adjuvant allows investigation of disease induction using specific autoantigens in several autoimmune diseases independently of toll‐like receptors, as well as optimization of vaccine delivery systems for infectious diseases. Curr. Protoc. Mouse Biol. 4:11‐24 © 2014 by John Wiley & Sons, Inc.

Keywords: polymer; poly‐N‐isopropylacrylamide; adjuvant; arthritis; collagen II

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Poly‐N‐Isopropylacrylamide (PNiPAAm) Synthesis
  • Basic Protocol 2: PNiPAAm‐CII Injection Preparation
  • Basic Protocol 3: Immunization Procedure for Induction of Chronic Arthritis
  • Basic Protocol 4: Clinical Evaluation of Arthritis
  • Basic Protocol 5: Histology of Arthritic Joints
  • Basic Protocol 6: Measurement of ANTI‐CII Antibody Levels
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Poly‐N‐Isopropylacrylamide (PNiPAAm) Synthesis

  Materials
  • N‐isopropylacrylamide (Sigma‐Aldrich)
  • Degassed water: keep distilled water on magnetic stirrer for 4 to 5 hr under vacuum in a sidearm flask connected to a vacuum pump
  • Nitrogen source
  • Ammonium persulfate (APS; Sigma‐Aldrich): 0.1% (w/v) in degassed water, as an initiator for reaction: check APS bottle for clumping (a sign of increased moisture content) before weighing; APS should always be kept dry, otherwise it will not be active during polymerization step (it is crucial to use freshly prepared APS solution for the reaction)
  • Tetramethylethylenediamine (TEMED; Sigma Aldrich): 0.1% (v/v) in degassed water as a catalyst for free radical reaction
  • Phosphate‐buffered saline (PBS; Life Technologies, cat. no. 10010‐056)
  • High‐Clarity polypropylene 50‐ml tubes (BD Falcon, cat. no. 352070)
  • 40°C water bath
  • Spatula

Basic Protocol 2: PNiPAAm‐CII Injection Preparation

  Materials
  • 1% (w/v) PNiPAAm ( protocol 1) in PBS
  • Collagen type II (CII) [can be prepared from several sources, e.g., rat chondrosarcoma, bovine nasal cartilage, chicken sternal cartilage, porcine cartilage, either in‐house using the standard protocol (Grab et al., ) or purchased from commercial sources, e.g., Sigma, Chondrex, or MD Biosciences; also see Rosloniec et al., ]
  • Freund's adjuvants: complete Freund adjuvant (CFA) and incomplete Freund adjuvant (IFA) (BD Difco)
  • Phosphate buffered saline (PBS; Life Technologies, cat. no. 10010‐056)
  • 15‐ml conical tubes (e.g., BD Falcon)
  • 1‐ml syringe and bent needle

Basic Protocol 3: Immunization Procedure for Induction of Chronic Arthritis

  Materials
  • Mice: e.g., QD = (B10.Q ×DBA/1) F1 mice (age, 8 to 12 weeks, sex, both; susceptibility of various inbred mouse strains and F1 intercrosses to this induction protocol is given in Table 13.2.2600)
  • PNiPAAm‐CII and CFA‐CII emulsions ( protocol 2) for immunization
  • Isoflurane/air mixture (3:1) as anesthesia
  • 70% ethanol
  • Incomplete Freund's adjuvant (IFA; BD Difco)
  • Phosphate‐buffered saline (PBS; Life Technologies, cat. no. 10010‐056)
  • Mouse cages placed in climate‐controlled environment with 12‐hr dark/light cycles
  • Wood shavings as bedding material
  • Standard mouse diet and water ad libitum
  • Ear puncher to make ear marks for identification of mice
  • 1‐ml syringe and 27‐G needles for intradermal immunization
  • Isoflurane/oxygen‐based anesthesia system fitted with an induction chamber and inhalation masks for mice
  • Additional reagents and equipment for intradermal injection of mice (Donovan and Brown, ) and blood collection from mice (Rathkolb et al., ).
Table 3.2.1   MaterialsCollagen Induced Arthritis Induced with CII and PNiPPAm Adjuvant in Inbred and F1 Mouse Strains (Shakya et al., ) a

Mouse strain Antigen + PNiPPAm Arthritis incidence (%) Maximum arthritis score (mean ± SEM)
B10.Q CII 7/25 (28) 18.4 ± 3
B10.P CII 0/25 (0) 0
B10.RIII CII 13/23 (57) 24 ± 4
BALB/c CII 0/15 (0) 0
C3H.Q CII 11/13 (85) 38 ± 6
C57Bl/6 CII 0/22 (0) 0
DBA/1 CII 13/26 (42) 21 ± 4
(BALB/c × B10.Q) F1 CII 4/19 (21) 34 ± 4
(B10.Q × DBA/1) F1 CII 13/18 (72) 41 ± 5
(B10.Q × DBA/1) F1 Ova 0/8 (0) 0
(B10.Q × DBA/1) F1 PBS 0/8 (0) 0

 aPNiPPAm, Poly‐N‐isopropylacrylamide; CII, collagen type II; Ova, ovalbumin; PBS, phosphate‐buffered saline; SEM, standard error of the mean.

Basic Protocol 4: Clinical Evaluation of Arthritis

  Materials
  • 4% (w/v) paraformaldehyde in PBS (Santa Cruz Biotechnology, cat no. NC0238527)
  • 50%, 70%, 80%, 95%, 100% (v/v) ethanol
  • Xylene
  • Decalcification solution (see recipe)
  • Surgical equipment including scalpels, scissors, and forceps
  • Tissue Tek VIP tissue processor (Sakura Finetek USA)
  • Additional reagents and equipment for euthanasia of mice (Donovan and Brown, ) and paraffin embedding and hematoxylin/eosin staining of tissues (Antal et al., )

Basic Protocol 5: Histology of Arthritic Joints

  Materials
  • Collagen type II (CII) [can be prepared from several sources, e.g., rat chondrosarcoma, bovine nasal cartilage, chicken sternal cartilage, porcine cartilage, either in‐house using the standard protocol (Grab et al., ) or purchased from commercial sources, e.g., Sigma, Chondrex, or MD Biosciences (also see Rosloniec et al., )]
  • 1× phosphate‐buffered saline (PBS; see recipe)
  • ELISA wash buffer (see recipe)
  • 5% (w/v) bovine serum albumin (Sigma‐Aldrich) in ELISA wash buffer
  • Pooled sera from arthritic mice or affinity purified polyclonal sera obtained from CII immunized mice as a positive control ( protocol 3).
  • Goat anti‐mouse IgG conjugated with horseradish peroxidase (Southern Biotech)
  • 2,2′‐azino‐bis(3‐ethylbenzothiazoline‐6‐sulfonic acid) (ABTS; Roche Diagnostic Systems)
  • ELISA plates (96‐well RIA/EIA plate)
  • ELISA plate reader (Synergy‐2, BioTek Instruments Inc.)
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Figures

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Literature Cited

Literature Cited
  Ahlqvist, E., Hultqvist, M., and Holmdahl, R. 2009. The value of animal models in predicting genetic susceptibility to complex diseases such as rheumatoid arthritis. Arthritis Res. Ther. 11:226.
  Antal, C., Muller, S., Wendling, O., Hérault, Y. and Mark, M. 2011. Standardized post‐mortem examination and fixation procedures for mutant and treated mice. Curr. Protoc. Mouse Biol. 1:17‐53.
  Bäcklund, J., Treschow, A., Bockermann, R., Holm, B., Holm, L., Issazadeh‐Navikas, S., Kihlberg, J., and Holmdahl, R. 2002. Glycosylation of type II collagen is of major importance for T cell tolerance and pathology in collagen‐induced arthritis. Eur. J. Immunol. 32:3776‐3784.
  Bevaart, L., Vervoordeldonk, M.J., and Tak, P.P. 2010. Collagen‐induced arthritis in mice. Methods Mol. Biol. 602:181‐192.
  Brand, D.D., Kang, A.H., and Rosloniec, E.F. 2003. Immunopathogenesis of collagen arthritis. Springer Semin, Immunopathol. 25:3‐18.
  Brand, D.D., Latham, K.A., and Rosloniec, E.F. 2007. Collagen‐induced arthritis. Nat. Protoc. 2:1269‐1275.
  Brunsberg, U., Gustafsson, K., Jansson, L., Michaëlsson, E., Ahrlund‐Richter, L., Pettersson, S., Mattsson, R., and Holmdahl R. 1994. Expression of a transgenic class II Ab gene confers susceptibility to collagen‐induced arthritis. Eur. J. Immunol. 24:1698‐1702.
  Burkhardt, H., Koller, T., Engström, A., Nandakumar, K.S., Turnay, J., Kraetsch, H.G., Kalden, J.R., and Holmdahl, R. 2002. Epitope‐specific recognition of type II collagen by rheumatoid arthritis antibodies is shared with recognition by antibodies that are arthritogenic in collagen‐induced arthritis in the mouse. Arthritis Rheum. 46:2339‐2348.
  Cathcart, E.S., Hayes, K.C., Gonnerman, W.A., Lazzari, A.A., and Franzblau, C. 1986. Experimental arthritis in a nonhuman primate. I. Induction by bovine type II collagen. Lab. Invest. 54:26‐31.
  Corten, C., Kretschmer K., and Kuckling, D. 2010. Novel multi‐responsive P2VP‐block‐PNiPAAm block copolymers via nitroxide mediated radical polymerization. Beilstein. J. Org. Chem. 6:756‐765.
  Corthay, A., Bäcklund, J., Broddefalk, J., Michaëlsson, E., Goldschmidt, T.J., Kihlberg, J., and Holmdahl, R. 1998. Epitope glycosylation plays a critical role for T cell recognition of type II collagen in collagen‐induced arthritis. Eur. J. Immunol. 28:2580‐2590.
  Courtenay, J.S., Dallman, M.J., Dayan, A.D., Martin, A., and Mosedale, B. 1980. Immunisation against heterologous type II collagen induces arthritis in mice. Nature 283:666‐668.
  Cremer, M.A., Rosloniec, E.F., and Kang, A.H. 1998. The cartilage collagens: A review of their structure, organization, and role in the pathogenesis of experimental arthritis in animals and in human rheumatic disease. J. Mol. Med. (Berl.) 76:275‐288.
  Donovan, J. and Brown, P. 2006a. Parenteral injections. Curr. Protoc. Immunol. 73:1.6.1‐1.6.10.
  Donovan, J. and Brown, P. 2006b. Euthanasia. Curr. Protoc. Immunol. 73:1.8.1‐1.8.4.
  Dzhambazov, B., Holmdahl, M., Yamada, H., Lu, S., Vestberg, M., Holm, B., Johnell, O., Kihlberg, J., and Holmdahl, R. 2005. The major T cell epitope on type II collagen is glycosylated in normal cartilage but modified by arthritis in both rats and humans. Eur. J. Immunol. 35:357‐366.
  Grab, B., Miles, A.J., Furcht, L.T., and Fields, G.B. 1996. Promotion of fibroblast adhesion by triple‐helical peptide models of type I collagen‐derived sequences. J. Biol. Chem. 271:12234‐12240.
  Holmdahl, R., Jansson, L., Gullberg, D., Rubin, K., Forsberg, P.O., and Klareskog, L. 1985. Incidence of arthritis and autoreactivity of anti‐collagen antibodies after immunization of DBA/1 mice with heterologous and autologous collagen II. Clin. Exp. Immunol. 62:639‐646.
  Holmdahl, R., Jansson, L., Larsson, E., Rubin, K., and Klareskog, L. 1986. Homologous type II collagen induces chronic and progressive arthritis in mice. Arthritis Rheum. 29:106‐113.
  Holmdahl, R., Andersson, M.E., Goldschmidt, T.J., Jansson, L., Karlsson, M., Malmström, V., and Mo, J. 1989. Collagen induced arthritis as an experimental model for rheumatoid arthritis. Immunogenetics, pathogenesis and autoimmunity. APMIS 97:575‐584.
  Holmdahl, R., Andersson, M., Goldschmidt, T.J., Gustafsson, K., Jansson, L., and Mo, J.A. 1990. Type II collagen autoimmunity in animals and provocations leading to arthritis. Immunol. Rev. 118:193‐232.
  Holmdahl, R., Carlsen, S., Mikulowska, A., Vestberg, M., Brunsberg, U., Hansson, A.S., Sundvall M., Jansson, L., and Pettersson, U. 1998. Genetic analysis of murine models for rheumatoid arthritis. In Human Genome Methods. (K.W. Adolpho, ed.) pp. 215‐238. CRC Press, New York.
  Kannan, K., Ortmann, R.A., and Kimpel, D. 2005. Animal models of rheumatoid arthritis and their relevance to human disease. Pathophysiology 12:167‐181.
  Kollias, G., Papadaki, P., Apparailly, F., Vervoordeldonk, M.J., Holmdahl, R., Baumans, V., Desaintes, C., Di Santo, J., Distler, J., Garside, P., Hegen, M., Huizinga, T.W., Jüngel, A., Klareskog, L., McInnes, I., Ragoussis, I., Schett, G., Hart, B., Tak, P.P., Toes, R., van den Berg, W., Wurst, W., and Gay, S. 2011. Animal models for arthritis: Innovative tools for prevention and therapy. Ann. Rheum. Dis. 70:1357‐1362.
  Lu, X., Zhang, L., Meng, L., and Liu, Y. 2007. Synthesis of poly(N‐isopropylacrylamide) by ATRP using a fluorescein‐based initiator. Polym. Bull. 59:195‐206.
  Luross, J.A. and Williams, N.A. 2001. Genetic and immunopathological processes underlying collagen‐induced arthritis. Immunology 103:407‐416.
  McInnes, I.B. and Schett, G. 2011. The pathogenesis of rheumatoid arthritis. N. Engl. J. Med. 365:2205‐2219.
  Monach, P.A., Benoist, C., and Mathis D. 2004. The role of antibodies in mouse models of rheumatoid arthritis, and relevance to human disease. Adv. Immunol. 82:217‐248.
  Myers, L.K., Rosloniec, E.F., Cremer, M.A., and Kang, A.H. 1997. Collagen‐induced arthritis, an animal model of autoimmunity. Life Sci. 61:1861‐1878.
  Nabozny, G.H., Baisch, J.M., Cheng, S., Cosgrove, D., Griffiths, M.M., Luthra, H.S., and David, C.S. 1996. HLA‐DQ8 transgenic mice are highly susceptible to collagen‐induced arthritis: A novel model for human polyarthritis. J. Exp. Med. 183:27‐37.
  Nandakumar, K.S. 2010. Pathogenic antibody recognition of cartilage. Cell Tissue Res. 339:213‐220.
  Nandakumar, K.S. 2012. Antibody mediated arthritis and new therapeutic avenues. In Antibody‐Mediated Drug Delivery Systems: Concepts, Technology, and Applications (Y. Pathak and S. Benita, eds.) pp. 407‐425. John Wiley & Sons Inc., Hoboken, New Jersey.
  Nandakumar, K.S. and Holmdahl, R. 2006. Antibody‐induced arthritis: Disease mechanisms and genes involved at the effector phase of arthritis. Arthritis Res. Ther. 8:223.
  Nandakumar, K.S. and Holmdahl, R. 2007. Collagen antibody induced arthritis. Arthritis Res. Methods Mol. Med. 136:215‐223.
  Rathkolb, B., Fuchs, H., Gailus‐Durner, V., Aigner, B., Wolf, E. and Hrabě de Angelis, M. 2013. Blood collection from mice and hematological analyses on mouse blood. Curr. Protoc. Mouse Biol. 3:101‐119.
  Rosloniec, E.F., Brand, D.D., Myers, L.K., Whittington, K.B., Gumanovskaya, M., Zaller, D.M., Woods, A., Altmann, D.M., Stuart, J.M., and Kang, A.H. 1997. An HLA‐DR1 transgene confers susceptibility to collagen‐induced arthritis elicited with human type II collagen. J. Exp. Med. 185:1113‐1122.
  Rosloniec, E.F., Cremer, M., Kang, A. H., Myers, L. K. and Brand, D.D. 2010. Collagen‐induced arthritis. Curr. Protoc. Immunol. 89:15.5.1‐15.5.25.
  Rowley, M.J., Nandakumar, K.S., and Holmdahl, R. 2008. The role of collagen antibodies in mediating arthritis. Mod. Rheumatol. 18:429‐441.
  Schulte, S., Unger, C., Mo, J.A., Wendler, O., Bauer, E., Frischholz, S., von der Mark, K., Kalden, J.R., Holmdahl, R., and Burkhardt, H. 1998. Arthritis‐related B cell epitopes in collagen II are conformation‐dependent and sterically privileged in accessible sites of cartilage collagen fibrils. J. Biol. Chem. 273:1551‐1561.
  Seki, N., Sudo, Y., Yoshioka, T., Sugihara, S., Fujitsu, T., Sakuma, S., Ogawa, T., Hamaoka, T., Senoh, H., and Fujiwara, H.1988. Type II collagen‐induced murine arthritis. I. Induction and perpetuation of arthritis require synergy between humoral and cell‐mediated immunity. J. Immunol. 140:1477‐1484.
  Shakya, A.K. and Nandakumar, K.S. 2013. Polymeric adjuvants in studying autoimmune responses and vaccination against infectious diseases. J. R. Soc. Interface 10:79.20120536.
  Shakya, A.K., Sami, H., Srivastava, A., and Kumar, A. 2010a. Stability of responsive polymer–protein bioconjugates. Prog. Polym. Sci. 35:459‐486.
  Shakya, A.K., Sharma, P., and Kumar, A. 2010b. Synthesis and characterization of thermo‐responsive poly(N‐isopropylacrylamide)‐bovine liver catalase bioconjugate. Enzyme Microb. Tech. 47:277‐282.
  Shakya, A.K., Kumar, A., and Nandakumar, K.S. 2011a. A thermo‐responsive polymeric [poly(N‐isopropylacrylamide)] adjuvant with collagen induced autoimmunity and arthritis in mice. J. R. Soc. Interface 8:1748‐1759.
  Shakya, A.K., Kumar, A., Klaczkowska, D., Hultqvist, M., Hagenow, K., Holmdahl, R., and Nandakumar, K.S. 2011b. Influence of MHC, T cells and oxidation status on arthritis induced with collagen and a thermo‐responsive polymeric adjuvant. Am. J. Pathol. 179:2490‐2500.
  Shakya, A.K., Holmdahl, R., Nandakumar, K.S., and Kumar, A. 2013. Characterization of chemically defined poly‐N‐isopropylacrylamide based copolymeric adjuvants. Vaccine 31:3519‐3527.
  Trentham, D.E., Townes, A.S., and Kang, A.H. 1977. Autoimmunity to type II collagen an experimental model of arthritis. J. Exp. Med. 146:857‐868.
  van Vollenhoven, R.F. 2013. Rheumatoid arthritis in 2012: Progress in RA genetics, pathology and therapy. Nat. Rev. Rheumatol. 9:70‐72.
  Vázquez‐Dorbatt, V., Tolstyka, Z.P., and Maynard, H.D. 2009. Synthesis of aminooxy end‐functionalized PNiPPAm by RAFT polymerization for protein and polysaccharide conjugation. Macromolecules 42:7650‐7656.
  Yoo, T.J., Kim, S‐Y., Stuart, J.M., Floyd, R.A., Olson, G.A., Cremer, M.A., and Kang, A.H. 1988. Induction of arthritis in monkeys by immunization with type II collagen. J. Exp. Med. 168:777‐782.
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