Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2013 May 30;8(5):e64888.
doi: 10.1371/journal.pone.0064888. Print 2013.

Active suppression induced by repetitive self-epitopes protects against EAE development

Fabiola Puentes  1 , Katharina Dickhaut, Maria Hofstätter, Kirsten Falk, Olaf Rötzschke
Affiliations

Active suppression induced by repetitive self-epitopes protects against EAE development

Fabiola Puentes et al. PLoS One. .

Abstract

Background: Autoimmune diseases result from a breakdown in self-tolerance to autoantigens. Self-tolerance is induced and sustained by central and peripheral mechanisms intended to deviate harmful immune responses and to maintain homeostasis, where regulatory T cells play a crucial role. The use of self-antigens in the study and treatment of a range of autoimmune diseases has been widely described; however, the mechanisms underlying the induced protection by these means are unclear. This study shows that protection of experimental autoimmune disease induced by T cell self-epitopes in a multimerized form (oligomers) is mediated by the induction of active suppression.

Principal findings: The experimental autoimmune encephalomyelitis (EAE) animal model for multiple sclerosis was used to study the mechanisms of protection induced by the treatment of oligomerized T cell epitope of myelin proteolipid protein (PLP139-151). Disease protection attained by the administration of oligomers was shown to be antigen specific and effective in both prevention and treatment of ongoing EAE. Oligomer mediated tolerance was actively transferred by cells from treated mice into adoptive hosts. The induction of active suppression was correlated with the recruitment of cells in the periphery associated with increased production of IL-10 and reduction of the pro-inflammatory cytokine TNF-α. The role of suppressive cytokines was demonstrated by the reversion of oligomer-induced protection after in vivo blocking of either IL-10 or TGF-β cytokines.

Conclusions: This study strongly supports an immunosuppressive role of repeat auto-antigens to control the development of EAE with potential applications in vaccination and antigen specific treatment of autoimmune diseases.

PubMed Disclaimer

Conflict of interest statement

Competing Interests: Dr. Olaf Rötzschke is an academic editor of PLOS ONE. No other conflicts of interest are to be reported by the authors. This does not alter the authors' adherence to all the PLOS ONE policies on sharing data and materials.

Figures

Figure 1
Figure 1. Protection against EAE development after administration of PLP139–151 16-mer.
(A) EAE was induced in SJL/J mice with 50 µg of the encephalitogenic PLP139–151 peptide emulsified in complete Freund's adjuvant containing Mycobacterium tuberculosis. Mice were untreated or treated intravenously with 50 µg of the PLP139–151 16-mer on day 7 after disease induction as represented by the arrow. (B) Mice were vaccinated subcutaneously or intravenously with 50 µg of PLP139–151 16-mer. Vaccination was performed 7 days before EAE induction as indicated by the arrow. Subcutaneous injection was given in incomplete adjuvant and for the intravenous injection the oligomer was dissolved in PBS. (C) Mice (n = 8) were treated individually once the first clinical signs of EAE appeared. 50 µg of the PLP139–151 16-mer was given intravenously in diseased mice. The plots show the mean ±SEM daily clinical score. Statistical significance between groups was determined was determined by Mann-Whitney U test. ***P<0.001, **P<0.01 and *P<0.05 compared with respective control group.
Figure 2
Figure 2. Antigen specificity is required for the protection induced by PLP139–151 16-mer.
EAE was induced in SJL/J mice with the PLP139–151 peptide in complete adjuvant. On day 7 after disease induction, mice were treated intravenously with PLP139–151 16-mer or with an unrelated oligomer containing an epitope derived of the influenza virus hemagglutinin protein (HA107–119 4-mer) as indicated by the arrow. Mice were monitored daily and the average ±SEM of the clinical score of five mice per group was calculated. Statistical significant difference between the PLP139–151 16-mer and HA107–119 4-mer treated groups was observed (**P<0.01).
Figure 3
Figure 3. Antigen specific stimulation and cytokine production of cells from PLP139–151 16-mer treated mice.
(A) Lymphoid tissue cells from oligomer treated mice were isolated on day 13 post-immunization and stimulated with titrated amounts of PLP139–151 peptide. 3×105 cells were incubated for 72 hours and the T cell response was measured by 3H-thymidine incorporation. Stimulation index was calculated as the ratio of mean counts per minute (cpm) from quadruplicate mice to the mean background cpm. An index >2 was considered positive. **P<0.01 and *P<0.05 compared to untreated controls by Student's t test analysis. (B) Ex vivo TNF-α secretion by cells from PLP139–151 16-mer treated and untreated mice was measured. Cells were isolated on day 6 after oligomer treatment and stimulated with 20 µg/ml PLP139–151 peptide and 4 µg/ml α-CD28 for 6 h. The intracellular TNF-α cytokine was quantified on CD4+CD154+ activated cells by FACS analysis. *P<0.05 compared to untreated controls by Student's t test analysis. (C) To measure IL-10 secretion, lymphoid tissue cells from PLP139–151 16-mer treated mice and untreated mice were stimulated on days 0 and 5 with different combinations of the PLP139–151 peptide (monomer) or the PLP139–151 16-mer (oligomer). Stimulation was performed with: (a) monomer-monomer, (b) monomer-oligomer, (c) oligomer-monomer and (d) oligomer-oligomer peptides. After 10 days in culture, supernatants were collected and the production of IL-10 was measured by ELISA. Results are expressed as the mean cytokine concentration (pg/ml). **P<0.01 compared to untreated controls by Student's t test analysis.
Figure 4
Figure 4. Effect of in vivo neutralization of IL-10 on the PLP139–151 16-mer treatment.
Oligomer treated mice were intraperitoneally injected with 0.5 mg of α-IL-10 or α-IL-10R (anti-IL-10 receptor). Injections were performed four times from the day of the treatment, every two days as indicated by the arrows. Untreated mice were used as control. Mice were monitored daily and the mean clinical score ±SEM was analyzed. These results are representative of two independent experiments. Statistical significance, **P<0.01 and *P<0.05 compared to PLP139–151 16-mer treated mice that were not administered anti-cytokine blocking antibodies. No effect was observed in oligomer treated mice given isotype control IgG1 antibody (Figure S2).
Figure 5
Figure 5. Effect of in vivo neutralization of TGF-β on the PLP139–151 16-mer treatment.
Oligomer treated mice were intraperitoneally injected with 0.5 mg of α-TGF-β or α-IL4. Injections were performed four times from the day of oligomer treatment every two days as indicated by the arrows. Clinical signs of EAE were scored daily. Data represent the mean ±SEM of the clinical score. **P<0.01 compared to PLP139–151 16-mer treated mice that were not administered anti-cytokine blocking antibodies. No effect was observed in oligomer treated mice injected with isotype control IgG1 antibody (Figure S2).
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Watanabe K, Sukumaran V, Veeraveedu PT, Thandavarayan RA, Gurusamy N, et al. (2011) Regulation of inflammation and myocardial fibrosis in experimental autoimmune myocarditis. Inflamm Allergy Drug Targets 10: 218–225. - PubMed
    1. van Herwijnen MJ, Wieten L, van der Zee R, van Kooten PJ, Wagenaar-Hilbers JP, et al. (2012) Regulatory T cells that recognize a ubiquitous stress-inducible self-antigen are long-lived suppressors of autoimmune arthritis. Proc Natl Acad Sci U S A 109: 14134–14139. - PMC - PubMed
    1. Liblau RS, Tisch R, Shokat K, Yang X, Dumont N, et al. (1996) Intravenous injection of soluble antigen induces thymic and peripheral T-cells apoptosis. Proc Natl Acad Sci U S A 93: 3031–3036. - PMC - PubMed
    1. Hogan SP, Foster PS, Charlton B, Slattery RM (1998) Prevention of Th2-mediated murine allergic airways disease by soluble antigen administration in the neonate. Proc Natl Acad Sci U S A 95: 2441–2445. - PMC - PubMed
    1. Liu Z, Harris PE, Colovai AI, Reed EF, Maffei A, et al. (1995) Suppression of the indirect pathway of T cell reactivity by high doses of allopeptide. Autoimmunity 21: 173–184. - PubMed

Publication types

MeSH terms

Grants and funding

This work was supported by a Deutsche Forschungsgemeinschaft Grant SFB650. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

LinkOut - more resources