Biochemically altered myelin triggers autoimmune demyelination

Abstract

Although immune attack against central nervous system (CNS) myelin is a central feature of multiple sclerosis (MS), its root cause is unresolved. In this report, we provide direct evidence that subtle biochemical modifications to brain myelin elicit pathological immune responses with radiological and histological properties similar to MS lesions. A subtle myelinopathy induced by abbreviated cuprizone treatment, coupled with subsequent immune stimulation, resulted in lesions of inflammatory demyelination. The degree of myelin injury dictated the resulting immune response; biochemical damage that was too limited or too extensive failed to trigger overt pathology. An inhibitor of peptidyl arginine deiminases (PADs), enzymes that alter myelin structure and correlate with MS lesion severity, mitigated pathology even when administered only during the myelin-altering phase. Moreover, cultured splenocytes were reactive against donor myelin isolates, a response that was substantially muted when splenocytes were exposed to myelin from donors treated with PAD inhibitors. By showing that a primary biochemical myelinopathy can trigger secondary pathological inflammation, "cuprizone autoimmune encephalitis" potentially reconciles conflicting theories about MS pathogenesis and provides a strong rationale for investigating myelin as a primary target for early, preventative therapy.

Keywords: citrullination; cuprizone; immunopathogenesis; multiple sclerosis; myelin.

Fig. 1.

Subdemyelinating cuprizone (CPZ) triggered Gd-enhancing lesions of inflammatory demyelination. (A) Brief CPZ in an induction phase was followed by immune boost (IB) and a 2-wk incubation period on regular chow. (B) Electron micrographs of myelin ultrastructure indicate that CPZ exposure was sufficiently brief as to alter but largely preserve myelin. (C) One-week post-IB, compared with naive, IB only, or CPZ only, only the combination of abbreviated CPZ then IB resulted in significant Gd enhancement (orange/red). (D) Two-weeks post-IB, compared with controls, CPZ+IB brains alone exhibited obvious T2 white matter lesions (white arrowheads; increased signal intensity on MRI) that corresponded to inflammatory demyelination, a reaction that was termed cuprizone autoimmune encephalitis (CAE). (E) Quantification of T1 relaxation-shortening effects of brain Gd, indicative of inflammation and blood–brain barrier leakage, were observed only in CPZ+IB. (F) T2w MRI reflected the severity and extent of CAE lesions, with cases that involved nearly 60% of the anterior–posterior extent of the corpus callosum. Each data point is an individual subject and bars represent SEM. EM, n = 6; Gd MRI, n = 4 naive; n = 6 IB only; n = 3 CPZ only; n = 8 CPZ then IB. Histology, n = 12 naive controls; n = 13 IB only; n = 24 CPZ only; n = 24 CPZ then IB. Significance was determined by one-way ANOVA. *P < 0.05, **P < 0.01, ****P < 0.0001. (Scale bars: B, 100 nm; D, Insets, 100 μm.)

Fig. 2.

The extent of myelin modifications modulated the resulting immune response. (A) Shorter or longer CPZ induction paradigms (1 and 3 wk) were followed, as in Fig. 1, by IB and a 2-wk incubation period. (B) Compared with IB alone, neither shorter (C) nor longer (E) CPZ induction periods resulted in significant Gd enhancement compared with an induction of 2 wk (D), previously shown to trigger inflammatory demyelination. At 2-wk post-IB, histopathology showed minimal inflammation in tissue sections from all conditions except those treated with 2 wk of CPZ followed by IB and 2 wk of incubation (2+IB+2; H). Namely, IB only (0+IB+2; F), 1-wk CPZ induction (1+IB+2; G), and 3-wk CPZ induction (3+IB+2; I) all contained significantly fewer CD45⁺ and Iba1⁺ cells. Inflammatory responses correlated with degree of LFB myelin loss (blue). The lone exception was the 3+IB+2 group, which was demyelinated as expected but not inflamed. (L) Noninflammatory myelin loss in 3+IB+2 was matched by inflammatory demyelination in 2+IB+2. [Scale bars: F, Upper, 10 μm; Lower, 100 μm (also apply to G–I).] Error bars represent SEM. Every data point in K and L is a distinct subject. n = 4–6 for all groups. Significance was determined by one-way ANOVA. **P < 0.01, ***P < 0.001, ****P < 0.0001; n/s, not significant.

Fig. 3.

Myelin citrullination was a major driver of CAE. (A) Without the small molecule PAD inhibitor KP-302, white matter T2 lesions (bright orange in top white box) in CAE (2+IB+2) brains were accompanied by inflammation and demyelination (Inset, blue/purple), and elevated CD45 and Iba1 fluorescence (green/red). (B) By contrast, KP-302 (50 mg/kg intraperitoneally, once daily) during both induction and incubation phases strongly mitigated myelin loss and reduced inflammation. (C) When administered during only the CPZ-induction phase, KP-302 was as effective as when delivered throughout both phases, reducing CD45/Iba1 immunofluorescence (D) and decreasing radiological lesion burden (E), in contrast to minimal protection afforded by the drug given postinduction only (SI Appendix, Fig. S5). (F) Western blot for citrullinated proteins at the end of the CPZ-induction phase demonstrated an up-regulation of citrullines particularly at MBP-relevant molecular weights. (G and H) Immunostaining showed degenerated myelin (QD9 in G) and increased citrullination (H and I) within callosal regions vulnerable to CAE. Error bars represent SEM. For each time point in F, n = 5 (SI Appendix, Fig. S4). Significance was determined by one-way ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; n/s, not significant. [Scale bars: A, Upper Insets, 100 μm; A, Lower 10 μm (scale bars in A also apply to B and C); G and G, Inset, 20 μm (scale bar in G also applies to H).]

Fig. 4.

CAE splenocytes reacted robustly and specifically to donor myelin. (A) Relative to splenocytes from naive controls without added antigens, cells derived from animals at the peak of CAE proliferated in response to myelin isolated from donor CPZ mouse brains. (B) CAE splenocytes were unreactive to myelin derived from CPZ animals treated in vivo with KP-302. (C) CAE splenocytes also reacted to naive myelin but not when the same naive donors were treated in vivo with KP-302. The duration of CPZ treatment—tied to varying degrees of citrullination (SI Appendix, Fig. S4)—modulated the resulting responses of CAE splenocytes. Each dot represents the mean of three technical antigen replicates from a single mouse in which n = 5 mice for all splenocyte sources and n = 3–4 mice for myelin isolates. Significance was determined by one-way ANOVA. *P < 0.05, **P < 0.01; n/s, not significant.

Fig. 5.

CAE elucidates a molecular pathway through which biochemically altered myelin triggers autoimmune demyelination in mouse. (A) With intact myelin, immune boost (IB) alone had little effect. (B) In stark contrast, an abbreviated, subdemyelinating cuprizone (CPZ) treatment induced a subtle biochemical myelinopathy [likely including citrullinated MBP (citMBP)] that, when followed by IB, secondarily triggered severe inflammatory demyelination. Such lesions are populated by citrullinated myelin-reactive innate [macrophages/microglia (Mφ/M)] and adaptive (T lymphocytes) immune cells, that drive severe demyelination and secondary and bystander axonal degeneration. (C) Paradoxically, IB administered after an overtly demyelinating 3-wk course of CPZ, with associated clearance of myelin antigens, had no effect. Only the combination of subtle biochemical myelin pathology together with appropriately timed immune stimulation, triggered brisk inflammatory demyelinating lesions similar to those found in MS.