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

  • 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

  • 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

  • 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

  • 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

  • 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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