Noncanonical autophagy in dendritic cells triggers CNS autoimmunity

ABSTRACT

Reactivation and expansion of myelin-reactive CD4⁺ T cells within the central nervous system (CNS) are considered to play a key role in the pathogenesis of multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE). We demonstrated that accumulation of myelin-specific CD4⁺ T cells within the CNS and subsequent clinical disease development require autophagy related (ATG) protein-dependent phagocytosis in dendritic cells (DCs). Genetic ablation of this pathway impairs presentation of myelin-associated antigen following phagocytosis of injured, phosphatidylserine-exposing oligodendroglial cells. Thus, DCs use ATG-dependent phagocytosis for enhanced presentation of myelin antigen, thereby linking oligodendrocyte injury with antigen processing and T cell-pathogenicity during autoimmune CNS inflammation.

KEYWORDS:

• autophagy
• EAE
• multiple sclerosis
• neuroinflammation
• phagocytosis

Phagocytosis constitutes a specialized mode of endocytosis, during which cells internalize solid extracellular cargo, and this process can be initiated through triggering of phagocytic receptors such as pattern recognition, opsonic, and phosphatidylserine (Ptd-L-ser) receptors. The emanating phagosome undergoes stepwise maturation and subsequently fuses with lysosomes followed by the enzymatic breakdown of the internalized constituents. In recent years, a novel pathway has been characterized that couples phagocytosis to certain autophagy-related (ATG) proteins. During this ATG-dependent phagocytosis, the ubiquitin-like autophagy protein LC3 is recruited to the outer membrane of single-membrane phagosomes. Whereas this cellular process is independent of the canonical macroautophagy core machinery, certain proteins such as ATG5 and ATG7 exert essential functions. ATG-dependent phagocytosis is involved in the promotion of lysosome-phagosome fusion, targeting of phagosomal cargo to pathogen recognition receptors and the provision of antigenic material to MHC class II-containing compartments. The relative importance of the aforementioned functions appears to be cell specific. Whereas ATG-dependent phagocytosis predominantly supports killing and degradation of engulfed pathogens in macrophages, in professional antigen-presenting cells such as DCs, ATG-dependent phagocytosis is employed to delay hydrolytic cleavage, which results in prolonged antigen storage and sustained antigen presentation via MHC class II.

MHC class II-mediated antigen presentation during CNS autoimmunity

Multiple sclerosis (MS) is a chronic immune-mediated disease of the CNS that develops in young adults. Akin to many autoimmune conditions, HLA-DR and -DQ alleles within the HLA class II region are the strongest risk-conferring genes. Whereas there is a general consensus that MHC class II molecules contribute to autoimmune disease risk by cognate interactions with autoreactive CD4⁺ T cells, how MHC class II-mediated antigen presentation regulates CD4⁺ T cell autoreactivity at the molecular level is still not fully understood. The emergence, activation and accumulation of myelin-reactive T cells within the CNS are thought to represent critical processes during the development and progression of MS, and encephalitogenic CD4⁺ T cells are the main driving forces in the animal model of MS, EAE. The adoptive transfer of primed myelin-specific CD4⁺ T cells into naïve recipient mice (adoptive transfer EAE, AT-EAE) models this effector phase, which is highly dependent on the presence of ITGAX/CD11c⁺ DCs within the CNS. How ITGAX/CD11c⁺ DCs initiate and sustain autoreactive CD4⁺ T cells within the CNS has not been fully elucidated, partly due to a lack of insight into the detailed antigen-processing route of how myelin self-antigens are processed and presented to CD4⁺ T cells.

Myelin-specific CD4⁺ T cells require ATG-dependent phagocytosis in DCs to induce EAE development

To address whether the autophagy machinery modulates EAE development, we generated conditional knockout mice (C57BL/6) for disruption of Atg5 in ITGAX/CD11c⁺ DCs. We identified a small population of CNS ITGAX/CD11c⁺ MHC class II^hi DCs that is targeted by Cre-mediated site-specific somatic recombination within the CNS. While ablation of ATG5 in ITGAX/CD11c⁺ DCs does not impair effector functions of CD4⁺ T cells nor alleviate peripheral priming of CD4⁺ T cells following active immunization with myelin protein, it diminishes accumulation of myelin-peptide-specific CD4⁺ T cells within the CNS and completely prevents clinical disease development following adoptive transfer of primed, encephalitogenic T cells.

Oligodendrocyte injury and concomitant focal demyelination constitute pathological hallmarks of MS lesions and during EAE development, and can even precede the formation of inflammatory infiltrates. Efficient local reactivation of myelin-specific CD4⁺ T cells in MS and EAE implies preceding phagocytosis, processing and presentation via MHC class II of oligodendrocyte-derived material. ATG-dependent phagocytosis can be initiated by engagement of Ptd-L-ser-recognizing receptors through injured target cells. We observed that, whereas ATG5 in ITGAX/CD11c⁺ DCs is negligible for the general capacity to phagocytose extracellular constituents, presentation of antigen derived from phagocytosed, Ptd-L-ser-exposing oligodendroglial cells is abrogated in the absence of ATG5.

Conclusions

Our data demonstrate that ATG-regulated phagocytosis of injured oligodendrocytes for MHC class II antigen presentation is critical for myelin-specific T cells to accumulate within the CNS and to permit EAE development (Figure 1). These findings link phagocytosis of oligodendrocyte-derived myelin with antigen processing and T cell-pathogenicity, and identify ATG-dependent phagocytosis in DCs as a key regulator in driving autoimmune CD4⁺ T cell-mediated CNS damage.

Figure 1. Schematic illustration summarizing how ATG5-dependent phagocytosis may be implicated in the provision of injured oligodendrocyte (ODC)-derived antigenic material to the MHC class II pathway in dendritic cells (DCs) during CNS autoimmunity. ATG5-dependent phagocytosis is initiated by the ingestion of Ptd-L-ser⁺ ODC fragments upon their binding of Ptd-L-ser receptors (Ptd-L-Ser-R). LC3-I is converted into LC3-II in an ATG5-dependent manner and recruited to the single membrane forming phagosome, which subsequently fuses with MHC class II-containing compartments (MIICs). Myelin-derived antigens are presented to encephalitogenic CD4⁺ T cells, thus facilitating the development and maintenance of neuroinflammation.

Disclosure of potential conflicts of interest

No potential conflicts of interest were disclosed.

Additional information

Funding

This work was supported by Deutsche Forschungsgemeinschaft [grant number KE 1831/1–1]; Novartis Stiftung für Medizinisch-Biologische Forschung; Schweizerische Multiple Sklerose Gesellschaft; Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung [grant number 31003A-169664].