ATG-Dependent Phagocytosis in Dendritic Cells Drives Myelin-Specific

ATG-dependent phagocytosis in dendritic cells drives PNAS PLUS + myelin-specific CD4 T cell pathogenicity during CNS inflammation Christian W. Kellera, Christina Sinaa,b, Monika B. Kotura, Giulia Ramellia, Sarah Mundtc, Isaak Quasta,d, Laure-Anne Ligeone, Patrick Webera, Burkhard Becherc, Christian Münze, and Jan D. Lünemanna,f,1 aInstitute of Experimental Immunology, Laboratory of Neuroinflammation, University of Zurich, 8057 Zurich, Switzerland; bBrain Research Institute, University of Zurich, 8057 Zurich, Switzerland; cInstitute of Experimental Immunology, Laboratory of Inflammation Research, University of Zurich, 8057 Zurich, Switzerland; dDepartment of Immunology and Pathology, Central Clinical School, Monash University, Melbourne, VIC 3004, Australia; eInstitute of Experimental Immunology, Laboratory of Viral Immunobiology, University of Zurich, 8057 Zurich, Switzerland; and fDepartment of Neurology, University Hospital Zurich, 8091 Zurich, Switzerland Edited by Lawrence Steinman, Stanford University School of Medicine, Stanford, CA, and approved November 10, 2017 (received for review August 2, 2017) Although reactivation and accumulation of autoreactive CD4+ T cells T cells recognize their target organ and become reactivated withintheCNSareconsideredtoplayakeyroleinthepathogenesis to induce sustained CNS tissue damage are, however, incom- of multiple sclerosis (MS) and its animal model, experimental auto- pletely understood. immune encephalomyelitis (EAE), the mechanisms of how these cells The autophagic machinery delivers cytoplasmic cargo and sub- recognize their target organ and induce sustained inflammation are strates for MHC class II presentation to late endosomes and lyso- incompletely understood. Here, we report that mice with conditional somes (10, 11). In addition, autophagy proteins regulate degradation deletion of the essential autophagy protein ATG5 in classical den- of extracellular material via the noncanonical use of autophagy- dritic cells (DCs), which are present at low frequencies in the non- related proteins (ATGs) during ATG8/microtubule-associated pro- diseased CNS, are completely resistant to EAE development tein 1A/1B light chain 3 (LC3)-associated phagocytosis (LAP) (12– following adoptive transfer of myelin-specific T cells and show sub- 18). Here, we report that DCs use ATG-dependent phagocytosis for + stantially reduced in situ CD4 T cell accumulation during the effector enhanced presentation of myelin antigen during autoimmune CNS INFLAMMATION phase of the disease. Endogenous myelin peptide presentation to inflammation, thereby linking oligodendrocyte injury with antigen IMMUNOLOGY AND CD4+ T cells following phagocytosis of injured, phosphatidylserine- processing and autoimmune T cell pathogenicity. exposing oligodendroglial cells is abrogated in the absence of ATG5. Pharmacological inhibition of ATG-dependent phagocytosis by the Results + + cardiac glycoside neriifolin, an inhibitor of the Na ,K -ATPase, de- Primed, Encephalitogenic CD4+ T Cells Require ATG5 Expression by lays the onset and reduces the clinical severity of EAE induced by DCs to Induce EAE. To address whether the autophagy machinery + myelin-specific CD4 T cells. These findings link phagocytosis of in- modulates EAE development, we generated conditional knock- + jured oligodendrocytes, a pathological hallmark of MS lesions and out mice (C57BL/6) for disruption of Atg5 in CD11c APCs − − during EAE, with myelin antigen processing and T cell pathogenicity, (Atg5flox/flox × CD11c-Cre-GFP, designated DC-Atg5 / ) (Fig. + and identify ATG-dependent phagocytosis in DCs as a key regulator 1A). Absence of ATG5 in CD11c cells led to complete pro- + in driving autoimmune CD4 T cell-mediated CNS damage. tection from EAE development upon adoptive transfer of T cell autophagy | neuroinflammation | EAE | multiple sclerosis Significance ultiple sclerosis (MS) is considered to be an antigen- How autoreactive CD4+ T cells recognize their target antigen and Mdriven, predominantly T cell-mediated autoimmune dis- induce sustained inflammation in organ-specific autoimmune ease of the CNS. Genome-wide association studies confirmed diseases is incompletely understood. In an experimental model that HLA class II haplotypes, in particular HLA-DRB1*1501/ of multiple sclerosis, we show that accumulation of myelin- + HLA-DRB5*0101, are the strongest genetic risk factors for MS specific CD4 T cells within the CNS and subsequent clinical dis- development (1, 2), a substantial fraction of T cells isolated from ease development requires autophagy protein (ATG)-dependent CNS lesional tissue and the cerebrospinal fluid from MS patients phagocytosis in dendritic cells (DCs). Absence of ATG-dependent are derived from clonal expansion (3–5), and inflammatory de- phagocytosis in DCs abrogates myelin presentation to CD4+ myelination in experimental autoimmune encephalomyelitis (EAE), T cells following phagocytosis of oligodendroglial cells, and its + an animal model for MS, is dependent on CD4 T cells that react pharmacological inhibition delays the onset and reduces the to myelin antigen (6, 7). clinical severity of experimental autoimmune encephalomyelitis. + Before infiltrating the CNS, autoreactive CD4 T cells are Thus, DCs use ATG-dependent phagocytosis for enhanced pre- primed in the peripheral immune system. Priming can be tar- sentation of myelin antigen during autoimmune CNS in- geted to skin draining lymph nodes after s.c. immunization with flammation, thereby linking oligodendrocyte injury with antigen myelin antigen and complete Freund’s adjuvant (CFA) during processing and autoimmune T cell pathogenicity. active EAE induction. In MS, the site where autoreactive T cells Author contributions: C.W.K., C.S., B.B., C.M., and J.D.L. designed research; C.W.K., C.S., are primed is not known, but the human disease is usually not M.B.K., G.R., S.M., I.Q., L.-A.L., and P.W. performed research; B.B. and C.M. contributed elicited by vaccinations and persists in the absence of any sys- new reagents/analytic tools; C.W.K., C.S., and M.B.K. analyzed data; and C.W.K. and J.D.L. temic inflammatory challenges. Local reactivation and sustained wrote the paper. accumulation of autoreactive T cells within the CNS are, The authors declare no conflict of interest. therefore, considered instrumental in both MS and EAE. This This article is a PNAS Direct Submission. effector phase can be modeled by adoptive transfer of primed Published under the PNAS license. + myelin-specific CD4 T cells into naïve mice (adoptive transfer 1To whom correspondence should be addressed. Email: [email protected]. + EAE; AT-EAE) and depends on the presence of CD11c + This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. antigen-presenting cells (APCs) (8, 9). How myelin-reactive CD4 1073/pnas.1713664114/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1713664114 PNAS Early Edition | 1of10 Downloaded by guest on October 2, 2021 – + neg +/+ −/− receptor-transgenic (TCR-tg) MOG35–55 specific CD4 T cells A CD11c / DC-Atg5 DC-Atg5 MOG CD11chi/ CD11b+/ derived from 2D2/TCR animals (Fig. 1B). Protection was MHCIIhi Ly6C+ also observed after adoptively transferring non–TCR-tg, poly- CD11chi MHCIIhi BMDCs + – ATG5- clonal encephalitogenic CD4 T cells obtained from MOG35–55 immunized C57BL/6 mice (Fig. 1C). In contrast, absence of 500 ATG12 + ATG5 in CD11c cells resulted in only minor and not statistically 400 LC3-I significant differences in incidence rates and clinical severity − − B cells CD4+ T cells 300 LC3-II grades in DC-Atg5 / mice upon active immunization (Fig. 1D). 200 % of Max CD11c-Cre-GFP mice were fully susceptible to EAE develop- 100 Actin Cre-GFP (MFI) Cre-GFP ment (Fig. S1A). + 0 Since loss of ATG5 in CD11c cells completely prevented the development of AT-EAE, we next profiled CD11c promoter- Cre-GFP + driven Cre-GFP expression in MHC class II CNS-resident CD11c+ CD11cneg Ly6C+ CD4+ APCs before EAE induction in naïve mice. A minor pop- + ATG5- ulation of CNS CD11b myeloid cells coexpressed high levels of ATG12 CD11c and MHC class II, indicative of classical DCs (cDCs), and Actin was efficiently targeted by Cre-mediated recombination (Fig. 2 A and B). By comparison, Cre-GFP expression in CD11cintMHC − − class IIhi cells derived from DC-Atg5 / mice was minor and not + + B DC-Atg5+/+ DC-Atg5−/− statistically different from DC-Atg5 / mice (Fig. S1B). CD11cneg 4 100 neg hi core myeloid populations such as CD11c Ly6C monocytes, pre- 80 cursors of CNS-invading monocyte-derived DCs and required for 3 the initiation of tissue inflammation in actively induced EAE EAE S l 60 (19–23), were not targeted by Cre-mediated recombination (Fig. ca 2 i n 2B), indicating that site-specific deletion of Atg5 was confined to 40 + + Cli Incidence (%) CD11c MHC class II DCs. However, we observed that up- 1 − − 20 regulation of MHC class II in DC-Atg5 / bone marrow-derived ean myeloid cells upon GM-CSF incubation precedes Cre-mediated M 0 0 1 10 20 30 recombination and loss of ATG5 (Fig. S1C), suggesting a days after transfer delayed targeting of ATG5 in monocyte-derived DCs in DC- −/− C Atg5 mice. 1.5 80 In C57BL/6 wild-type mice, ATG5 protein expression was core + detectable in CNS-derived CD11c cells in naïve mice as well as − − 60 after induction of AT-EAE (Fig. 2C). In DC-Atg5 / mice, fre- hi hi EAE1.0 S l quencies of CD11c MHC class II CNS DCs were similar +/+ ca 40 i compared with DC-Atg5 littermates (Fig. 2D). Moreover, n hi hi 0.5 ATG5-deficient CNS-derived CD11c MHC class II DCs did Incidence (%) Cli 20 not differ from their ATG5-competent counterparts in expres- ean sion levels of MHC class II, CD40 and CD86 (Fig. 2E). Micro- M 0 0 1 10 20 30 glial cells, in which expression of CD11c and MHC class II can days after transfer be induced upon activation, did not express CD11c, exhibited no detectable levels of MHC class II molecules (Fig. 2 F and G), and were observed at similar frequencies in naïve mice when D − − + + 3 100 comparing DC-Atg5 / with DC-Atg5 / (Fig. 2H). Furthermore, core + ns frequencies of splenic DC and monocyte subsets, CD4 T cells, 80 + + + CD8 T cells, and CD4 Foxp3 regulatory T cells and B cells EAE S 2 l 60 (Fig.

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