Neutralizing IL-17 protects the optic nerve from autoimmune pathology and prevents retinal nerve fiber layer atrophy during experimental autoimmune encephalomyelitis
Introduction
Experimental autoimmune encephalomyelitis (EAE) is an inflammatory demyelinating disease of the central nervous system (CNS). It can be induced by active immunization with myelin antigens and is the most frequently used animal model to study multiple sclerosis (MS). Originally, MS and EAE were thought to be Th1 mediated diseases because IFN-γ, the signature cytokine of Th1 cells, was found in EAE and MS lesions. However, the “Th1 paradigm” was challenged when it was found that IL-12p35 deficient mice and IFN-γ deficient mice were not resistant to EAE but developed even enhanced disease. Yet, IL-12p40 KO mice are resistant to EAE and so are IL-23p19 KO animals. In fact, IL-12 and IL-23 are heterodimeric cytokines the share a common p40 subunit but have private p35 (IL-12) and p19 (IL-23) subunits, respectively. Although the role of IL-23 in vivo is not entirely clear yet, it stabilizes the phenotype of Th17 cells that produce IL-17A (IL-17) and IL-17F as their signature cytokines [1]. Many recent findings support the notion that Th17 cells are major drivers of autoimmune pathology in EAE and MS [2], [3], [4].
Predominance of either Th1 or Th17 cells has an impact on the pattern and localization of inflammatory demyelination in the CNS. Adoptive transfer of IL-23 polarized Th17 cells into naïve hosts caused neutrophil rich lesions with increased tissue expression of CXCL-1 and CXCL-2 whereas transfer of IL-12 polarized Th1 cells resulted in high CXCL-9 and CXCL-10 expression within the CNS inducing macrophage rich cellular infiltrates [5]. Also, the ratio of Th17 to Th1 cells seems to dictate the localization of lesions in the CNS. In a model, in which the ratio of Th17 vs Th1 was controlled by the affinity for different MHC class II complexes, high Th17/Th1 ratios resulted in brain inflammation while low Th17/Th1 ratios, i. e. a preponderance of Th1 cells, promoted spinal cord inflammation [6]. Notably, Th17 cells are able to infiltrate the brain parenchyma in the absence of VLA-4 expression while infiltration of the spinal cord by encephalitogenic Th1 cells or Th17 cells that re-express IFN-γ is dependent on VLA-4 [7]. Thus, different parts of the CNS might constitute different “immunologic” compartments.
Although optic neuritis is a very common inflammatory manifestation of MS, immunologic mechanisms affecting the optic nerve during EAE have not been studied in detail. Here, we characterize murine optic neuritis during EAE and establish spectral domain optical coherence tomography (SD-OCT) as an appropriate method to study optic neuritis in a longitudinal manner. We show that IL-17 is a crucial cytokine for the development of murine optic neuritis and promotes axonal atrophy in the optic nerve. In summary, our data suggest that the optic nerve might be an immunologic compartment distinct from the spinal cord.
Section snippets
Animals, induction of EAE, and neutralization of IL-17
EAE was induced in mice on pure C57BL/6 background by subcutaneous (s.c.) application of an emulsion containing 200 μg MOG35–55 peptide (MEVGWYRSPFSRVVHLYRNGK) and 250 μg Mycobacterium tuberculosis H37Ra (Difco) in Freund's adjuvant oil (CFA) plus intravenous (i.v.) injection of 200 ng pertussis toxin (Sigma–Aldrich) on days 0 and 2. Mice were rated in a blinded manner according to an EAE score from 0 to 5 (0 = no signs of disease; 1 = tail paresis; 2 = unsteady gate; 3 = paraplegia;
Population dynamics of immune cell infiltration into different CNS compartments during EAE
In order to compare immune cell infiltrates in the brain, spinal cord, and optic nerve during MOG35–55 induced EAE in a quantitative manner, we dissected these compartments and prepared individual single cell suspensions followed by flow cytometric analysis. The onset of leukocyte infiltration into the optic nerve was on day 9 after immunization and was delayed by about one day as compared with the spinal cord. Immune cell infiltration in the optic nerve reached its peak at about day 16. At the
Discussion
In this study, we show that autoimmune inflammation in the optic nerve is characterized by a Th17 type response resulting in RNFL and GCL+ atrophy as measured by SD-OCT. While autoimmune pathology in the spinal cord is only partly inhibited by anti-IL-17 treatment, neutralization of IL-17 is sufficient to completely prevent structural damage to the optic nerve.
Several methods of non-invasive assessment of rodent murine optic neuritis have been reported: Shindler et al. examined pupillary light
Conclusion
SD-OCT is an appropriate technique to study optic neuritis in a longitudinal manner during EAE in vivo. By using OCT in EAE, we identified superior responses of the optic nerve compartment to IL-17 neutralizing therapy suggesting that the optic nerve – in contrast to the spinal cord – is particularly prone to support IL-17 mediated inflammatory responses during CNS autoimmunity. Further studies will have to address whether neutralization of IL-17 by antibody treatment only reduces inflammatory
Acknowledgments
We thank Franco di Padova and Frank Kolbinger from Novartis (Basel, Switzerland) for providing the rat anti-mouse IL-17 antibody. We thank Veronika Husterer and Svenja Woeste for expert technical assistance. T.K. was supported by grants of the Deutsche Forschungsgemeinschaft (SFB 1054, TR128, and SyNergy) and intramural funding of the Technische Universität München (KKF). B.H. was supported by a grant of the DFG (TR128). All authors declare no competing financial interests.
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