ATG-dependent phagocytosis in dendritic cells drives PNAS PLUS + myelin-specific CD4 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 September 27, 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. S2A), MHC class II and costimulatory molecule expression + ca i on splenic cDCs (Fig. S2B), and CD4 T cell inflammatory cy-

n 40 1 tokine expression (IFNγ, IL-17, and GM-CSF) upon activation Incidence (%) Cli − − 20 were unchanged in DC-Atg5 / mice compared with their DC- +/+ ean Atg5 control littermates (Fig. S2C). Using the identical

M 0 0 1 10 20 30 CD11c-Cre and Atg5flox/flox strains for DC-specific deletion of days after immunization Atg5, Lee et al. (11) previously demonstrated that Atg5-deficient

− − DCs show intact migratory capacities, similar expression levels of Fig. 1. DC-Atg5 / mice are resistant to EAE induced by primed, myelin- specific T cells. (A) Representative histograms depicting Cre-GFP expression + + + + of either CD11c MHCII splenic DCs (cDCs), CD11cnegCD11b Ly6C mono- + + + − − cytes, B cells, or CD4 T cells in DC-Atg5 / (black) or DC-Atg5 / (red) mice in steady state (Upper Left). Quantification of Cre-GFP median fluorescence Quantification of disease incidence is shown (Right). (C) EAE was induced via + + − − intensity (MFI; DC-Atg5 / , n = 7; DC-Atg5 / , n = 9) (Upper Middle). Western adoptively transferring C57BL/6 wild type-derived encephalitogenic CD4+ + + − − blot analysis for protein expression of the ATG5–ATG12 complex, LC3-I, and T cells into DC-Atg5 / or DC-Atg5 / mice. Each data point represents the + LC3-II in CD11c BMDCs (Upper Right) and protein expression of the ATG5– mean of seven animals (Left). One representative of two independent ex- + + ATG12 complex in primary splenic cell populations CD11c , CD11cnegLy6C , periments is shown. Quantification of disease incidence is shown (Right). + +/+ −/− and CD4 (Lower) derived from DC-Atg5 or DC-Atg5 mice is depicted. (D) EAE was induced via active immunization with MOG35–55 peptide in DC- Actin served as a loading control. One representative of two experiments is Atg5+/+ or DC-Atg5−/− mice. Each data point represents the mean of 15 or shown. (B) EAE was induced via adoptively transferring 2D2/TCRMOG-derived more animals. Pooled data of three independent experiments are shown + + + − − encephalitogenic CD4 T cells into DC-Atg5 / (black triangles) or DC-Atg5 / (Left). Quantification of disease incidence is shown (Right). Statistical anal- mice (red circles). Each data point represents the mean of 44 or more ani- ysis: Mean ± SEM is depicted. Two-way ANOVA (D, Left) was applied. ns, not mals. Pooled data of eight independent experiments are shown (Left). significant: P > 0.05.

2of10 | www.pnas.org/cgi/doi/10.1073/pnas.1713664114 Keller et al. Downloaded by guest on September 27, 2021 + PNAS PLUS MHC class II, CD40, and CD86 in the steady state, and upon AT-EAE induction (Fig. S3). In line with the pivotal role of CD4 + + immune activation along with similar secretion levels of IL- T cells in EAE (24, 25), the proportion of CD4 but not CD8 + + 12p40, IL-6, and TNF-α. T cells positively correlated with the severity of EAE in DC-Atg5 / Thus, a minor population of CD11chiMHC class IIhi DCs animals (Fig. S4). These data indicate that protection from EAE − − within the nondiseased CNS is targeted by Cre-mediated Atg5 development in DC-Atg5 / mice is associated with reduced ac- − − + deletion in DC-Atg5 / mice. ATG5 deficiency does not affect tivation and accumulation of CD4 T cells within the CNS. their frequency or expression levels of MHC class II and cos- Since the overall frequencies of CNS-infiltrating leukocytes timulatory molecules, but prevents EAE development induced producing proinflammatory upon ex vivo restimulation + −/− by primed, myelin-specific CD4 T cells. with MOG35–55 were significantly reduced in DC-Atg5 com- + + pared with DC-Atg5 / mice at the peak of disease (Fig. 3C), we + + ATG5 in DCs Does Not Impair Priming of Myelin-Specific CD4 T Cells next determined the ability of CNS-invading CD4 T cells to but Regulates Their Accumulation Within the CNS. We next de- produce proinflammatory cytokines. To this end, leukocytes termined frequencies and effector functions of CNS-infiltrating were isolated and purified from the CNS at the peak of disease − − + + T cells in DC-Atg5 / compared with DC-Atg5 / littermates. To (day 21 ± 1). For each animal the CNS-derived cell suspension this end, mice were killed on day 21 (±1) upon adoptive transfer was divided into two groups, and cells were restimulated for 4 h – + of MOG35–55 specific CD4 T cells, at the peak of disease in either with MOG35–55 peptide or OVA323–339 peptide. Pro- + + − − + + + + DC-Atg5 / animals. DC-Atg5 / mice showed substantially duction of IFNγ , IL-17 , and GM-CSF by CNS CD4 T cells + + −/− lower proportions of CNS-infiltrating CD4 but not CD8 upon rechallenge with MOG35–55 was preserved in DC-Atg5 T cells (Fig. 3A). In addition, frequencies of activated CNS mice (Fig. 3 D and E). − − CD44hi T cells were markedly reduced in DC-Atg5 / mice (Fig. To additionally determine whether ATG5 deficiency in DCs + + − − 3B). Reduced accumulation of MOG-specific CD4 T cells was impairs CD4 T cell priming, we immunized DC-Atg5 / mice + + also observed at an earlier time point (day 12 post transfer) after and DC-Atg5 / littermates with full-length MOG protein/CFA

CD11cneg/ A B CD11chi/MHCIIhi CD11b+/Ly6C+ CD4+ T cells 33.1 63.4 INFLAMMATION IMMUNOLOGY AND 75 CD45 % of Max % of Max % of Max FSC-H SSC-A

Cre-GFP FSC-A FSC-A dead and Ly6G 2000 2000 2000 1500 1500 1500 94.4 5.37 12.6 1000 1000 1000 500 500 500 Cre-GFP (MFI) Cre-GFP 0 0 0 SSC-A CD45

94.1 CD11c CD11c+ CD11cneg/Ly6C+ 86.3 ATG5- ATG5- DC-Atg5+/+ ATG12 ATG12 / CD11b CD11b MHCII DC-Atg5− − Actin Actin n=6 n=6 n=6 n=6 C57BL/6 WT CD11chi/MHCIIhi * C EF20000 * d0 d14 ATG5- 15000 ATG12 ns Actin 10000 % of Max % of Max n=9 n=6 % of Max

CD11c (MFI) CD11c 5000

MHCII CD86 CD40 0 hi hi D CD11c /MHCII Microglia CD11chi/MHCIIhi ns ns ns ns ns ns ns GH500 + 100 15 1.0 5000 800 400

+ 400 80 0.8 4000 600 300

300 /CD11b 60

10 + 0.6 3000 400 200 200 40

2000 MHCII (MFI)

0.4 CD40 (MFI) CD86 (MFI) 5 MHCII (MFI) 100 20 % of CD11b % of % of parent 200 100 0.2 1000 0 0 0 0 0 0 0 Microglia % of CD45 Microglia

Fig. 2. CD11c+ Cre-GFP–expressing DCs in the CNS before EAE induction. (A) Gating strategy for flow cytometry analysis in the CNS of naïve DC-Atg5+/+ and − − + DC-Atg5 / mice. First, single leukocytes were defined by applying the respective gates (leukocytes: SSC-A vs. FSC-A; single cells: FSC-H vs. FSC-A). Next, CD45 + + + cells were gated on, while excluding Ly6G neutrophils and dead cells. After gating on CD11b myeloid cells, microglia were defined as CD45loCD11b . Inside + + + the CD45hiCD11b cell gate, it was further gated on CD11chiMHCIIhi cells. Within the remainder of the cells it was subgated on CD11cnegCD11b Ly6C monocytes (not shown). (B) Representative histograms (Upper) depicting Cre-GFP expression of either CNS-resident CD11chiMHCIIhi cells (Upper Left) or CNS- derived CD11cnegCD11b+Ly6C+ monocytes (Upper Middle) in naïve mice. Representative histogram depicting Cre-GFP expression in CNS-infiltrating CD4+ T cells at the peak of disease (day 21 ± 1) (Upper Right). Quantification of Cre-GFP MFI is depicted (Middle). Western blot analysis for protein expression of the ATG5–ATG12 complex in CNS-derived CD11c+ cells (Lower Left) and CD11cnegLy6C+ cells (Lower Right) (DC-Atg5+/+, n = 6; DC-Atg5−/−, n = 6). Actin served as a + loading control. (C) Western blot analysis for protein expression of the ATG5–ATG12 complex in CNS-derived CD11c cells of C57BL/6 wild-type (WT) mice before induction of adoptive transfer EAE (n = 9) or on day 14 after induction of adoptive transfer EAE (n = 6). Actin served as a loading control. + + + (D) Quantified frequencies (percentage of parent, Left; percentage of CD11b cells, Right) of CNS-resident CD11chiMHCIIhi cells in naïve DC-Atg5 / and − − DC-Atg5 / mice. (E) Representative histograms (Upper) depicting either MHCII (Left), CD86 (Middle), or CD40 (Right) MFI of CNS-resident CD11chiMHCIIhi cells. Quantification of MFI values is shown (Lower). (F) Microglia versus CD11chiMHCIIhi cell phenotypes in DC-Atg5−/− mice before EAE induction. CD45lo/intCD11b+ microglial cells do not express CD11c to a substantial level at steady state compared with CD11chiMHCIIhi CNS cells. (G) Surface expression levels of MHCII are + + − − + − − similar on microglia when comparing DC-Atg5 / and DC-Atg5 / mice. (H) Frequencies of CD45lo/intCD11b microglial cells are unchanged in DC-Atg5 / mice. + + − − + + Pooled data of two independent experiments are shown (DC-Atg5 / , n = 5andDC-Atg5 / , n = 5 for naïve myeloid compartments; DC-Atg5 / , n = 10 and − − + DC-Atg5 / , n = 12 for peak of disease CD4 T cell analysis). Statistical analysis: Mean ± SEM is depicted. Unpaired two-tailed Student t test was applied. ns, not significant: P > 0.05; *P < 0.05.

Keller et al. PNAS Early Edition | 3of10 Downloaded by guest on September 27, 2021 DC-Atg5+/+ DC-Atg5−/− Thus, ATG5 in DCs is not required for priming myelin-specific A 100 + 80 *** CD4 T cells upon active immunization. Lack of ATG5 in DCs does not affect the encephalitogenic capacity of primed, CNS- +/+ 60 70.1 13.3 + DC-Atg5 infiltrating CD4 T cells, but restrains their in situ reactivation −/− DC-Atg5 of live single cells 40 SSC-A

+ and accumulation. 20 0

% CD45 Absence of ATG5 in DCs Abrogates Endogenous Myelin Peptide CD45 50 50 Presentation Following Phagocytosis of Injured Oligodendroglial ** ns

cells Cells. Canonical autophagy delivers intracellular antigens for cells 40

40 + + 23.7 8.03 MHC class II presentation, while EAE development is driven by 30 30 an antigen not intrinsically expressed by professional APCs and CD4

of CD45 2.78 of CD45 20 20 10.1 + + therefore requires endocytosis, followed by myelin antigen pro- 10 10 cessing and presentation. ATGs may contribute to extracellular % CD8 % CD4 0 0 antigen processing through phagosome maturation, regulated CD8 through cytosolic attachment of ATG8/LC3 in a process called B +/+ CD4+ CD8+ DC-Atg5 DC-Atg5−/− ATG-dependent or LC3-associated phagocytosis. We therefore 80 20 −/− *** *** 58.2 17.8 determined whether Atg5 DCs are impaired in phagocytosis + + + 60 15 and/or in their capability to present myelin antigen for CD4 40 10 T cell activation. First, we assessed general phagocytosis by loading CD44 + % CD44 % CD44 CD11c bone marrow-derived DCs (BMDCs) with polystyrene 20 5 beads decorated with and without MOG1–125 full-length protein and 0 0 CD4 beads decorated with the LAP-triggering Toll-like receptor C DE* (TLR)2 agonist Pam3CSK4 (26). Ingested beads were quantified ** −/− 0.8 30 ns ns – ** OVA MOG by laser scanning confocal microscopy. Both DC-Atg5 and 323-339 35-55 +/+ 0.6 20 – + + DC-Atg5 derived BMDCs phagocytosed similar numbers of

0.4 T cells (%) DC- Atg5 + IFN IFN 10 either naked polystyrene beads, beads that had been decorated 0.2 with MOG protein, and beads decorated with Pam3CSK4 (Fig. 0 of CD4 0 of live single cells (%) MOG O M 35-55 1.31 7.79 5A), indicating that the general capacity of ATG5-deficient DCs +/+ 1.0 6 * to phagocytose extracellular material is not compromised.

+ ** + 0.8 ** ns ns 0.6 4 ATG-dependent phagocytosis of extracellular material re- T cells (%) IL-17A IL-17A 0.4 + 2 quires triggering through receptor-mediated antigen uptake such

0.2 GM-CSF

DC- Atg5 as phosphatidylserine (Ptd-L-Ser)-recognizing receptors, danger- 0 of CD4 0 of live single cells (%) MOG O M 35-55 associated molecular pattern (DAMP) receptors, TLR1/2, TLR2/6, 1.2 30 * * ns

+ 3.67 7.37 + 1.0 ** TLR4, TLR9, and Dectin-1, or Fc receptors recognizing DNA

ns −

20 / − immune complexes (12, 14, 17, 18, 27). Ptd-L-Ser can be exposed T cells (%)

0.5 + GM-CSF GM-CSF 10 on membrane debris derived from damaged cells or specifically CD4

0 of CD4 0 flipped to the outer cell-membrane leaflet during apoptosis (28). of live single cells (%) MOG 35-55 O M Oligodendrocyte injury and concomitant focal demyelination + Fig. 3. Lack of ATG5 in CD11c DCs limits accumulation of encephalitogenic constitute unique pathological hallmarks of MS lesions and during + CD4 T cells within the CNS. (A) Twenty-one days (±1) after adoptive transfer EAE development (29, 30), and can even precede the formation EAE induction, DC-Atg5−/− mice exhibit significantly fewer CD45+ infiltrates of inflammatory infiltrates (31–33). We therefore hypothesized in the CNS. Furthermore, quantification of immune cell subsets shows sig- that uptake of damaged Ptd-L-Ser–exposing oligodendroglial cells + − − + nificantly lower proportions of CD4 T cells in DC-Atg5 / compared with DC- by CD11c DCs triggers myelin-specific T cell activation in an + + Atg5 / mice at the peak of disease in the CNS, whereas no difference is + ATG5-dependent manner. observed in the CD8 T cell compartment. (B) At the peak of disease, DC- hi − − + + To test our hypothesis, irradiated Ptd-L-Ser compared with Atg5 / mice exhibit significantly lower frequencies of activated CD44 /CD4 lo + + nonirradiated Ptd-L-Ser MOG-expressing oligodendroglial cells − − + + and CD8 T cells in the CNS. (C) The ability of CNS-invading CD4 T cells to (Fig. 5 B–D) were loaded onto DC-Atg5 / – or DC-Atg5 / –derived produce proinflammatory cytokines was determined. Leukocytes were iso- ± primary splenic DCs. Endogenous myelin-derived peptide lated and purified from the CNS at the peak of disease (day 21 1). For each γ animal the CNS-derived cell suspension was divided into two groups, and cells presentation was assessed by IFN production of cocultured – + γ were restimulated for 4 h with either MOG35–55 peptide (M) or OVA323–339 MOG35–55 specific CD4 T cells. T cell IFN production upon + − − peptide (O). CNS-infiltrating CD4 T cells in DC-Atg5 / mice maintain their presentation of endogenous myelin antigen derived from Ptd-L- capacity to produce effector cytokines. DC-Atg5+/+ mice contain significantly Serhi oligodendroglial cells was abrogated in ATG5-deficient DCs – more (percentage of live single cells) -producing MOG35–55 specific (Fig. 5E). ATG5-dependent activation of myelin-specific T cells CD4+ T cells in the CNS than DC-Atg−/− mice. (D)BothDC-Atg5−/−– and DC- + + + required processing of oligodendroglial cell components exposing Atg5 / –derived CD4 T cells are capable of secreting cytokines (IFNγ, IL-17A, −/− +/+ Ptd-L-Ser, since loading of DC-Atg5 – or DC-Atg5 –derived and GM-CSF) upon restimulation with MOG35–55 (but not with OVA323–339)to + + DCs with MOG protein-decorated polystyrene beads resulted in similar degrees. (E) Representative density plot for GM-CSF CD4 T cells in −/− +/+ similar T cell proliferation upon coculture with myelin-specific the CNS of DC-Atg5 or DC-Atg5 mice at the peak of disease. Pooled data + of two independent experiments are shown. Statistical analysis: Mean is CD4 Tcells(Fig.5F). These data indicate that recognition and depicted. Unpaired two-tailed Student t test was applied. ns, not significant: engulfment of injured oligodendroglial cells by DCs induce myelin P > 0.05; *P < 0.05, **P < 0.01, ***P < 0.001. peptide presentation on MHC class II molecules and subsequent + activation of primed myelin-specific CD4 T cells through ATG5- regulated phagocytosis (Fig. 6). (or full-length OVA protein/CFA) and restimulated splenic + CD4 T cells with titrated numbers of C57BL/6 wild type- The Cardiac Glycoside Neriifolin Inhibits ATG-Dependent Phagocytosis, derived peptide-pulsed DCs 7 d after immunization (Fig. 4A). Delays Onset, and Reduces Clinical Severity of EAE. Although several Antigen-specific proliferation and IFNγ cytokine production of interventions are available to inhibit autophagy at the nucleation, + − − CD4 T cells primed in DC-Atg5 / mice were similar to those elongation, fusion, or degradation phase (34), pharmacological + + primed in DC-Atg5 / littermates (Fig. 4B). inhibitors of ATG-dependent phagocytosis have not been identified

4of10 | www.pnas.org/cgi/doi/10.1073/pnas.1713664114 Keller et al. Downloaded by guest on September 27, 2021 PNAS PLUS A D0 D7 D11 + CFSE/ELISA Immunization with full length MOG1-125 or Isolation of CD4 T cells OVA 1-385 C57BL/6 WT Co-culture Pulsed with MOG35-55 or OVA323-339

splenic CD11c+ DCs DC-Atg5+/+ DC-Atg5−/− DC-Atg5+/+ DC-Atg5−/−

B MOG-primed MOG-primed Non-primed CD4+ T cells CD4+ T cells CD4+ T cells 40 2500 +/+

dim DC-Atg5 DC-Atg5−/− 2000 30 ns ns 1500 ns ns ns

CFSE 20 ns [pg/ml] + 1000 10 IFN 500 DL DL % CD4 0 0 0 1×105 2×105 4×105 0 1×105 2×105 4×105 0 5 5 5 1×10 2×10 4×10

MOG35-55-pulsed DCs MOG35-55-pulsed DCs OVA-primed OVA-primed Non-primed + + + CD4 T cells CD4 T cells CD4 T cells INFLAMMATION IMMUNOLOGY AND 40 ns 2500

dim ns ns ns ns 30 2000 ns 1500 CFSE

+ 20 [pg/ml] 1000 10 IFN 500 DL DL % CD4 0 0 5 5 0 1×105 2×105 4×105 0 1×105 2×105 4×105 0 5 1×10 4×10 2×10

OVA323-339-pulsed DCs OVA323-339-pulsed DCs

Fig. 4. Loss of ATG5 in DCs does not impair priming of antigen-specific CD4+ Tcells.(A) Experimental setup. (B) Quantification of DC-Atg5−/−– and DC-Atg5+/+–derived + CD4 T cell proliferation via carboxyfluorescein succinimidyl ester (CFSE) dilution in the presence of increasing amounts of wild type-derived peptide- + pulsed DCs (Upper and Lower Left). CD4 T cell response (IFNγ secretion) is unchanged upon coculture with peptide-pulsed DCs (Upper and Lower Right). Pooled data of two independent experiments are shown. Statistical analysis: Mean ± SEM is depicted. Unpaired two-tailed Student t test was applied. ns, not significant: P > 0.05. DL, detection limit.

so far. The autophagy protein beclin 1/ATG6 initiates autophago- which are known to carry out high levels of ATG-dependent some formation via interaction with the class III type phospho- phagocytosis (12, 16). In line with the aforementioned data inositide 3-kinase (PI3K3)/Vps34. Vps34, along with its regulatory (38), LC3-II levels were decreased upon 24 h of neriifolin protein kinase Vps15, is a critical regulator of endocytic sorting in treatment compared with the vehicle control (Fig. 7A). Next, we yeast and mammalian cells (35), and both kinases form a hetero- investigated whether neriifolin inhibits ATG-dependent phago- trimeric complex along with beclin 1, referred to as the “beclin 1– cytosis. Recruitment of LC3 to zymosan-containing phagosomes, Vps34 complex” (36). This complex, in addition to its role in ini- a hallmark of ATG-dependent phagocytosis (16), was signifi- tiating autophagosome formation, was shown to be recruited to cantly reduced by exposure of RAW 264.7 cells to neriifolin (Fig. phagosomes, responsible for phagosomal maturation (37), and 7 B and C). The compound did not exhibit any cytotoxicity at the suggested to mediate LC3 lipidation of phagosomal membranes range of concentration tested (0.1 to 1,000 nM) (Fig. 7D). In during LAP (16). vivo, neriifolin treatment significantly delayed the onset and re- The CNS-penetrating cardiac glycoside neriifolin, an inhibitor duced disease severity of EAE upon active immunization and + + + of the Na ,K -ATPase, a plasma membrane pump that gener- following transfer of myelin-specific CD4 T cells (Fig. 7E). + + ates Na and K gradients across the membrane, has recently been identified to inhibit ATG-dependent autophagy processes Discussion + through interaction with beclin 1 (38). In rats, treatment with Our study shows that myelin-specific CD4 T cells require ATG- neriifolin prevented the increase in autophagy after hypoxia– dependent phagocytosis in DCs to induce sustained inflamma- + ischemia injury as measured by decreased levels of LC3 lipidation tion and EAE development. CD11c cells within the CNS alone, (38). We first tested if exposure to neriifolin regulates auto- that is, in the absence of secondary lymphoid tissues, are suffi- phagic activity by virtue of LC3 lipidation in RAW 264.7 cells, cient to present antigen in vivo to primed myelin-reactive T cells

Keller et al. PNAS Early Edition | 5of10 Downloaded by guest on September 27, 2021 to mediate CNS inflammation (8, 9, 39). In the steady state, A + + 100 ns ns ns CD11c MHC class II DCs within the CNS are enriched in the 80 DC-Atg5+/+ choroid plexus (40, 41) which, along with the meningeal vascu- DC-Atg5−/− lature, is an active site for immune trafficking into and out of the 60 CNS (42–44) and a first port of entry for pathogenic T cells 40 during EAE (45). Choroid plexus DCs resemble splenic cDCs in morphology, gene expression profile, antigen-presenting func- 20 % Cells with beads tion, and their shared intrinsic requirement for Fms-related ty- 0 rosine kinase (Flt)3 ligand (46). We identified a small population uncoated MOG Pam CSK 1-125 3 4 of CD11chiMHC class IIhi DCs that are specifically targeted by -coated Cre-mediated recombination within the nondiseased CNS. Tar- + BDWT ODC C geted deletion of ATG5 in these cells abrogated CD4 T cell MOG-transduced untreated accumulation and completely prevented clinical disease devel- MOG+ (= ODC ) irradiated 60 opment following adoptive transfer of primed, encephalitogenic ** T cells, which reflects the effector phase of EAE. Following 50 active EAE induction with s.c. immunization with antigen/CFA, differences in incidence rates and clinical severity grades in DC- 40 − − Atg5 / compared with control mice were minor and not statis- 30 tically significant. These data indicate distinct functions of % of Max % of Max 20 ATG5 in cDCs in active vs. AT-EAE. While reactivation of encephalitogenic T cells within the CNS is required in both in-

% Ptd-L-Ser-expressing 10 MOG Ptd-L-Ser lo hi duction protocols, they differ in their requirement for cDCs for neg hi + ODCMOG+ disease initiation. CD11c Ly6C CCR2 monocytes, precur- E +/+ sors of CNS-invading monocyte-derived DCs, but not cDCs, are 1600 DC-Atg5 DC-Atg5−/− required for the initiation of tissue inflammation in actively in- duced EAE (19–23). Studies over the past years that employed 1000 ** inducible ablation of cDCs through CD11c promoter-driven 600 human diphtheria toxin (DT) receptor (DTR) expression addi- + 600 tionally investigated whether CD11c cells are required for T cell priming during EAE development following active immuniza- tion. Isaksson et al. (22) reported that DT-treated mice still 400 develop EAE after active immunization, indicating that a pop-

IFN γ (pg/ml) ulation of APCs other than cDCs executed initial priming of encephalitogenic T cells. However, DT treatment did not ablate + 200 CD11c APCs in the CNS during the peak of disease, and + CD11c APCs present in the CNS still could present myelin to invading encephalitogenic T cells (22). Yogev et al. (23) also 0 used CD11c-DTR mice to deplete cDCs including dermal DCs, lo hi and found that mice lacking these DC populations are even 35-55 Ptd-L-Ser hypersusceptible to EAE induction, supporting the concept that α− CD3/ − CD28

2D2 only DCs maintain peripheral tolerance. However, they demonstrated F MOG 100 ns ns ns ns ns ns ns ns that after T cell priming and during T cell invasion into the CNS (effector phase), DC-less mice showed reduced clinical scores compared with control mice. Along these lines, adoptive transfer 80 + + dim of primed, encephalitogenic CD4 T cells into CD11c -depleted recipients leads to dramatically reduced disease incidence and scores 60 (9). Thus, while the aforementioned studies reflect the complexity of CFSE

+ APCs during T cell priming and indicate that, after active EAE in- 40 duction, priming synapses can be formed between non–cDC-APC + populations such as inflammatory CD11cnegLy6ChiCCR2 mono- % CD4 cytes (19, 20), they do not contradict but support the concept that 20 + CD11c cDCs are the most efficient APCs in driving the reactivation

0 0.5:1 0.5:1 1:1 2:1 4:1 8:1

35-55 uncoated MOG -coated 1-125 shown. (E) Coculture of MOG-specific 2D2/TCRMOG-derived CD4+ T cells with α− CD3/ − CD28 + hi splenic CD11c DCs that had previously been pulsed with either Ptd-L-Ser – MOG lo MOG+ + or Ptd-L-Ser –expressing ODC . CD4 T cell response (IFNγ secretion) is + MOG+ hi Fig. 5. Endogenous myelin presentation by CD11c DCs is abrogated in the augmented upon coculture with ODC -pulsed Ptd-L-Ser DCs. Absence − − + + + absence of ATG5. (A) DC-Atg5 / – and DC-Atg5 / –derived BMDCs were in- of ATG5 in DCs abrogates CD4 T cell response upon coculture with MOG+ hi cubated for 4 h with either uncoated, MOG1–125–coated, or Pam3CSK4– ODC -pulsed Ptd-L-Ser DCs. One representative of >3 independent coated polystyrene beads, and the percentage of bead-containing cells was experiments is shown. (F) Coculture of 2D2/TCRMOG-derived CD4+ with DC- − − + + quantified via confocal microscopy. (B) Representative histogram comparing Atg5 / – and DC-Atg5 / –derived FAC-sorted splenic DCs in the presence of MOG+ + MOG-transduced oligodendroglial cell line MO3.13 (ODC ) with wild- MOG1–125–coated beads. Different bead:DC ratios are depicted. CD4 Tcell + type MO3.13 cells (WT ODC) for surface MOG expression. (C) ODCMOG proliferation was quantified via CFSE dilution. Pooled data of three in- were either UVB–irradiated (870 mJ/cm2) or left untreated, which resulted in dependent experiments are shown. Statistical analysis: Mean ± SEM is hi lo MOG+ Ptd-L-Ser – and Ptd-L-Ser –expressing ODC .(D) Quantification via depicted. Unpaired two-tailed Student t test was applied. ns, not significant: annexin V staining. Pooled data of three independent experiments are P > 0.05; **P < 0.01.

6of10 | www.pnas.org/cgi/doi/10.1073/pnas.1713664114 Keller et al. Downloaded by guest on September 27, 2021 differences in incidence rates and clinical severity grades in DC- PNAS PLUS −/− T Atg5 mice upon active immunization. Bhattacharya et al. (48) previously reported that CD11c-Cre–driven deletion of ATG7 T LC3 T ameliorates actively induced EAE. Different efficacies in targeting T T ATG5 ATG7 in inflammatory monocyte and monocyte-derived DC T T Ptd-L-Ser-R T subsets, which were not analyzed in the aforementioned study, T might have contributed to the more distinctive phenotype during Phagosome TCR T actively induced EAE compared with our study. Treatment with MHCII T T neriifolin, which inhibits ATG-dependent phagocytosis and does Injured ODC DC Myelin-specific EAE not specifically target either inflammatory monocytes and their (Ptd-L-Ser+) CD4+ T cells progeny or cDCs, ameliorated both active and adoptive transfer EAE, indicating that ATG-dependent phagocytosis contributes to Fig. 6. Provision of injured oligodendrocyte-derived antigenic material via both disease models. ATG5-dependent phagocytosis. Schematic depiction summarizing how ATG5- In addition to cDCs, myeloid cells within the CNS include dependent phagocytosis contributes to the provision of injured oligoden- parenchymal microglia and nonparenchymal meningeal, peri- drocyte (ODC)-derived antigenic material to the MHC class II antigen pre- vascular, and choroid-plexus (49). Assessing the + sentation pathway in CNS DCs. Parts of compromised, Ptd-L-Ser ODCs are precise immunological and antigen-presenting functions of the phagocytosed upon ligation of Ptd-L-Ser receptors (Ptd-L-Ser-Rs) triggering aforementioned subsets located at CNS–periphery interfaces in ATG5-dependent phagocytosis. LC3-I is converted into LC3-II in an ATG5- vivo is still hampered by their scarcity and the dearth of specific dependent manner and recruited to the single-membrane phagosome, methods of isolation and targeting (49). While we found Cre- which fuses with MHC class II-containing compartments. Myelin-derived an- + mediated site-specific deletion of Atg5 to be confined to CD11c MHC tigenic material will be subsequently presented to encephalitogenic T cells, + facilitating the development and maintenance of neuroinflammation. class II DCs, leptomeningeal, perivascular-space, and choroid- plexus myeloid cells might have potential antigen presentation

+ capacity during the course of EAE. Whether ATG-dependent of primed myelin-specific CD4 T cells during the effector phase of phagocytosis in these subsets additionally contributes to myelin- EAE (47). + + specific CD4 T cell reactivation and EAE development remains Lack of Cre-mediated recombination in CD11cnegLy6C mono- to be clarified. INFLAMMATION

cytes and the delayed targeting of ATG5 in CNS-invading monocyte- Both ATG5 and ATG7 are part of the ubiquitin-like protein IMMUNOLOGY AND derived DCs might have contributed to the nonsignificant and minor conjugation system that mediates LC3 lipidation, required for

Zymosan + Neriifolin AB CZymosan + Vehicle inert beads + Neriifolin Vehicle Neriifolin LC3 Zymosan DAPI Merge inert beads + Vehicle LC3-II 15 Actin * *** CQ − + − +

Vehicle 10 4

3 Vehicle 5 2 Neriifolin LAPosomes/picture +

1 LC3 Neriifolin LC3II / actin / LC3II ratio 0 0 020406080 CQ − − + + time [min] 2.0 4 D Immortalized macrophages RAW 264.7 E ** 80 30 min 6 h 24 h 80 30 min 6 h 24 h 1.5 3 60 60 Vehicle 1.0 2 40 40 Neriifolin 0.5 1 20 20 Staurosporine 0 0 0 0 VSN VSN VSN VSN VSN V N S 05 Days post-onset 2.0 4 + % cells dead Dendritic cells CD4 T cells B cells ** 60 30 min 6 h 24 h 20 30 min 6 h 24 h 30 30 min 6 h 24 h 1.5 3 Mean Clinical EAE Score Mean Clinical EAE Score 50 15 20 1.0 2 40 10 10 0.5 1 30 5 20 0 0 0 0 05 VSN VSN VSN V N S V N SVSN V N S V N SVSN Vehicle Days post-onset Neriifolin

Fig. 7. Cardiac glycoside neriifolin inhibits ATG-dependent phagocytosis and delays onset of EAE. (A) Western blot analysis for protein expression of LC3-II in RAW 264.7 cells in the absence (vehicle, 0.5% ethanol in PBS) or presence of neriifolin (1 μM). Actin served as a loading control. One representative of eight independent experiments (Upper) and quantification of Western blot analyses (Lower) are shown. (B) Immunofluorescence confocal laser scanning micros- copy of RAW 264.7 cells (neriifolin, 1 μM; vehicle, 0.5% ethanol in PBS) visualizing LC3 (green), zymosan (red), and DAPI (blue). Representative pictures from the 80-min time point are depicted. Original magnification with 63×, 1.4 N.A. oil immersion lens. White arrows indicate LC3-decorated, zymosan-containing LAPosomes. (Scale bars, 5 μm.) (C) Quantification and kinetics of zymosan-triggered LAPosome formation in the absence (vehicle, 0.5% ethanol in PBS) or presence of neriifolin (1 μM). Non–LAP-triggering inert polystyrene beads were used as a control. (D) FACS analysis of neriifolin cytotoxicity using myeloid cell + + + + lines and primary (splenic) CD11chiMHCIIhi dendritic cells, CD3 CD4 T cells, and CD19 MHCII B cells. Cells were exposed to increasing amounts of neriifolin (N; 0.1, 1, 10, and 1,000 nM) for either 30 min, 6 h, or 24 h. The protein kinase inhibitor staurosporine (S; 1 μM) was used as a positive and vehicle (V; 0.5% ethanol in PBS) as a negative control. (E) Neriifolin treatment (0.25 mg/kg; vehicle, 0.5% ethanol in PBS) delays onset and ameliorates disease severity of adoptive transfer (Upper) and active (Lower) EAE. Pooled data of two independent experiments are depicted. Each dot represents one individual animal. Statistical analysis: Mean ± SEM is depicted. Unpaired two-tailed Student t test was applied. *P < 0.05, **P < 0.01, ***P < 0.001. CQ, chloroquine.

Keller et al. PNAS Early Edition | 7of10 Downloaded by guest on September 27, 2021 Table 1. Primers for genotyping Primer Source Description Sequence, 5′-3′

Atg5flox/flox primer 1 (63) exon3-1 GAATATGAAGGCACACCCCTGAAATG Atg5flox/flox primer 2 (63) short2 GTACTGCATAATGGTTTAACTCTTGC Atg5flox/flox primer 3 (63) check2 ACAACGTCGAGCACAGCTGCGCAAGG Atg5flox/flox primer 4 (63) 5L2 CAGGGAATGGTGTCTCCCAC CD11c-Cre primer 1 oIMR1084 Jackson Transgene forward GCGGTCTGGCAGTAAAAACTATC CD11c-Cre primer 2 oIMR1085 Jackson Transgene reverse GTGAAACAGCATTGCTGTCACTT CD11c-Cre primer 3 oIMR7338 Jackson Internal positive control forward CTAGGCCACAGAATTGAAAGATCT CD11c-Cre primer 4 oIMR7339 Jackson Internal positive control reverse GTAGGTGGAAATTCTAGCATCATCC

+ + the formation of double-membrane autophagosomes and the associated disease conditions (57). We found that the Na ,K- decoration of single-membrane phagosomes following extracel- ATPase inhibitor neriifolin, reported to target beclin 1 (38), inhibits lular antigen uptake during ATG-dependent phagocytosis (12– the formation of LC3-associated phagosomes and ameliorates EAE 14, 16). It has previously been suggested that loss of ATG7 in development. In addition to its previously reported inhibitory effect + DCs reduces in vivo priming of myelin-specific CD4 T cells on hypoxia-induced LC3 lipidation in vivo (38), neriifolin might following MOG/CFA immunization (48). The conclusion made exert autophagy-independent, including neuroprotective, functions in the aforementioned study is based on the finding that sple- (58), as multiple chemical agents that are currently available to − − nocytes derived from MOG-immunized DC-Atg7 / mice pro- activate or inhibit autophagy have limited specificity for the auto- duce less IL-2 5 to 6 d following restimulation with MOG phagic process. Consistent with our findings, treatment with chlo- peptide compared with ATG7-proficient mice (48). While these roquine, which inhibits degradation of autolysosomes, before EAE − − data indicate a reduced recall response in DC-Atg7 / mice upon onset was reported to delay disease progression and attenuate its T cell reactivation, they do not specifically address the capability severity (48). While the aforementioned effects might not be ex- + of ATG7-deficient DCs to prime CD4 T cells. We demonstrate clusively mediated through ATG-dependent phagocytosis and an- that absence of ATG5 in DCs does not impair their ability to tigen presentation, they support the concept that pharmacological + prime myelin-specific CD4 T cells in vivo. Instead, absence of inhibition of the autophagy machinery in DCs should be further ATG5 in DCs abrogates endogenous myelin peptide pre- explored for its potential therapeutic merit to limit autoimmune + + sentation to already-primed myelin-specific CD4 T cells fol- CD4 T cell-mediated CNS inflammation. + lowing phagocytosis of injured Ptd-L-Ser oligodendroglial cells, Absence of ATG-dependent phagocytosis in myeloid cells by indicating that ATG-dependent phagocytosis of extracellular lysozyme M-Cre–mediated gene deletion, which targets macro- antigen contributes to MHC class II presentation but requires phages, monocytes, some neutrophils, and cDCs, was recently receptor-mediated antigen uptake (12, 14, 17, 26, 27). shown to lead to development of a systemic autoinflammatory + CNS-infiltrating, myelin-specific CD4 T cells require de novo syndrome in mice with increased expression of IFN signature phagocytosis and processing from intact myelin to induce sus- genes, occurrence of anti–double-stranded DNA and nuclear tained demyelination (8, 39, 50, 51). Similar to its function antibodies, and signs of kidney damage, commonly associated during canonical autophagy, ATG5 forms a heterodimer with with systemic lupus erythematosus (18). The aforementioned ATG12 that is bound to ATG16L1 to produce the ATG5– study showed that macrophages deficient in ATG7 or Rubicon, ATG12–ATG16L1 complex (52). The E3 ligase activity of this both required for ATG-dependent phagocytosis, did engulf, but complex finally mediates LC3 lipidation at the phagosomal not effectively clear, dying cells and produced proinflammatory membrane of late phagosomes (12, 14, 18, 53). Thus, closure of cytokines, including IL-1β and IL-6, upon challenge with apo- − − the plasma membrane around extracellular cargo and early ptotic cell material (18). An increased propensity of ATG7 / phagosome formation are upstream of LC3 lipidation and in- macrophages to produce proinflammatory cytokines after en- dependent of ATG5 (Fig. 6). In line with these data, we found gulfment of apoptotic cells has also been described in vitro (14), that the general capacity of ATG5-deficient DCs to phagocytose indicating that defective ATG-dependent phagocytosis in mac- extracellular material is not compromised. In contrast, their ca- rophages results in a failure to digest engulfed dying cells, + pacity to stimulate myelin-specific CD4 Tcellsfollowingpro- leading to elevated inflammatory cytokine production and the −/− cessing of damaged Ptd-L-Ser–exposing oligodendroglial cells was development of a lupus-like syndrome. In our study, DC-Atg5 + greatly diminished. Although exposure of Ptd-L-Ser is primarily mice were protected from the development of a CD4 T cell- associated with nonimmunogenic clearance of apoptotic cells, it mediated autoimmune disease, and ATG5 was required for DCs also occurs during nonapoptotic cell death, where externalized to efficiently present antigen derived from injured oligoden- + Ptd-L-Ser can be attributed to rupture of the plasma membrane droglial cells to myelin-specific CD4 T cells. We find our data in rather than being an active exposure process, and DAMPs re- line with in vitro studies that identified ATG-dependent leased from damaged cells can additionally activate inflammatory phagocytosis in professional APCs supporting downstream + programs (54). DAMP receptors additionally identified to trigger CD4 T cell responses by promoting sustained MHC class II ATG-dependent phagocytosis include TLRs such as TLR2 and antigen presentation (26, 27). ATG-dependent phagocytosis/ TLR9, whose genetic deletion ameliorates EAE development LAP was shown to be required for retinoid recycling by phago- induced by myelin-specific T cells, that is, in the absence of any cytic retinal pigment epithelial cells, which contributes to main- apparent microbial involvement (55, 56). While the receptors and taining vision in mice (13), and to occur on macropinosomes (15) signaling pathways involved remain to be identified, our data in- and at the ruffled border in osteoclasts (59), suggesting that this dicate that ATG-dependent phagocytosis of injured oligoden- pathway contributes significantly to physiology in multiple con- + + drocytes critically augments CD4 T cell pathogenicity during texts (60). During CD4 T cell-driven CNS inflammation, DCs autoimmune CNS demyelination. use ATG5 for enhanced presentation of endocytosed cargo de- Autophagosome formation, maturation, and lysosomal degra- rived from damaged oligodendrocytes on MHC class II mole- dation can be targeted by pharmacological compounds whose cules, thus linking oligodendrocyte injury with myelin antigen therapeutic efficacies could be demonstrated in various autophagy- processing and T cell pathogenicity.

8of10 | www.pnas.org/cgi/doi/10.1073/pnas.1713664114 Keller et al. Downloaded by guest on September 27, 2021 Loss of oligodendrocytes is a distinctive feature in tissue adja- leukocytes were purified. Bulk leukocytes were cultured in R10 medium PNAS PLUS cent to rapidly expanding MS lesions (61), and can precede the supplemented with recombinant IL-23 (10 ng/mL; 14-8231-63; eBioscience), μ formation of inflammatory infiltrates (31–33). Moreover, epi- MOG35–55 (10 g/mL), and 1% penicillin/streptomycin (P/S) for 48 h at 37 °C MOG × 6 × 6 genomic changes in genes affecting oligodendrocyte susceptibility and 5% CO2.Bulk2D2/TCR cells (10 10 ) or wild-type cells (10 to 15 10 ) were injected i.p. into each recipient mouse, which had been sublethally to damage and decreased expression of genes regulating oligo- irradiated with 550 rad (RS 2000; Rad Source Technologies) 1 d before cell dendrocyte survival were recently detected in pathology-free areas transfer. Clinical manifestations of EAE and weight loss were monitored and of MS-affected brains (62), compatible with the concept that oli- documented daily. Mice were scored as follows: 0, no detectable signs of godendrocyte injury might trigger or augment myelin protein EAE; 0.5, distal limp tail; 1, complete limp tail; 1.5, limp tail and hindlimb processing if professional APCs are present. Future immunohis- weakness; 2, unilateral partial hindlimb paralysis; 2.5, bilateral partial hind- tochemical studies will determine whether ATG-dependent limb paralysis; 3, complete bilateral hindlimb paralysis; 3.5, complete bi- phagocytosis can be visualized in APCs associated with MS le- lateral hindlimb paralysis and partial forelimb paralysis; 4, moribund (animal sions. We conclude from our data that ATG-regulated phagocy- unable to move due to paralysis); and 5, animal found dead. In the following tosis of injured oligodendrocytes for antigen presentation is instances, animals were immediately euthanized with CO2 upon evaluation: required for encephalitogenic T cells to induce sustained CNS disease score of 3 for more than 7 d, disease score of 3.5 for more than 3 d, inflammation and disease development in mice. ATG-regulated and reaching disease score of 4. The last documented score of euthanized or dead animals was carried forward for statistical analysis. phagocytosis in DCs might also be relevant for the perpetuation of MOG+ neuroinflammation in patients with MS and, therefore, a potential Ptd-L-Ser–Expressing Cell Phagocytosis and Coculture Assay. ODC were − − therapeutic target to limit inflammatory CNS damage. either UVB-irradiated (870 mJ/cm2) or left untreated. DC-Atg5 / – and DC- + + + Atg5 / –derived splenocytes were isolated, and the frequency of CD11c + Materials and Methods MHCII DCs was determined via FACS using a small aliquot of the splenocyte − − + + Mice. Wild-type C57BL/6 mice were purchased from Janvier Labs. Congenic suspension. Bulk splenocytes from DC-Atg5 / and DC-Atg5 / mice were

C57BL/6-CD45.1 mice were purchased from Charles River Laboratories. cocultured overnight (37 °C, 5% CO2) in cell-culture dishes with either Ptd-L- flox/flox hi lo MOG+ MOG+ + + Atg5 mice were a kind gift of Noboru Mizushima, University of Tokyo, Ser or Ptd-L-Ser ODC (10:1 ratio of ODC to CD11c MHCII DCs Tokyo, Japan (63). Tg(Itgax-cre,-EGFP)4097Ach mice designated CD11c-Cre based on their frequencies within the splenocyte suspension determined were purchased from Jackson Laboratory. MOG-specific TCR transgenic earlier) in R10 medium supplemented with 1% P/S. The next day, CD11c+MHCII+ MOG mice C57BL/6-Tg(Tcra2D2,Tcrb2D2)1Kuch/J (designated 2D2/TCR ) were a DCs were magnetic activated cell sorting (MACS)-purified from all condi- −/− lo MOG+ −/− hi MOG+ kind gift from Vijay K. Kuchroo, Harvard Institutes of Medicine, Boston, MA. tions (DC-Atg5 + Ptd-L-Ser ODC ; DC-Atg5 + Ptd-L-Ser ODC ; MOG +/+ lo MOG+ +/+ hi MOG+ 2D2/TCR mice were backcrossed to CD45.1 congenic C57BL/6 mice in our DC-Atg5 + Ptd-L-Ser ODC ;DC-Atg5 + Ptd-L-Ser ODC ). CD11c-

facility. All animals were bred and housed in the University of Zurich animal enriched fractions were further cocultured at a 1:5 ratio (DC:T cell) in a INFLAMMATION MOG IMMUNOLOGY AND facility in individually ventilated cages on a 12-h light/dark cycle with food 96-well U-bottom plate overnight (37 °C, 5% CO2)with2D2/TCR - + and water available ad libitum according to institutional guidelines and Swiss derived MACS-purified CD4 T cells in a total volume of 200 μL flox/flox animal laws. CD11c-Cre mice were crossed with Atg5 mice to obtain R10 supplemented with 1% P/S and IL-2 (10 ng/mL, 402-ML-100; R&D − − CD11c-Cre × Atg5flox/flox mice (designated DC-Atg5 / ) on a C57BL/6 back- MOG + Systems). Wells containing 2D2/TCR CD4 Tcellsonly,MOG35–55 flox/flox +/+ ground. Atg5 mice (designated DC-Atg5 ) were used as littermate peptide (20 μg/mL), or anti-CD3 and anti-CD28 antibodies (5 μg/mL each) controls. All animal protocols were approved by and conducted in accordance were also included. All conditions were performed in triplicate. After 24 h of with the veterinary office of the canton of Zurich (protocol ZH210/2014). incubation, cell-culture supernatants were collected for analysis of IFNγ con- centration by ELISA (88-7314-76; eBioscience). Genotyping. CD11c-Cre and Atg5flox/flox mouse genotypes were determined via PCR analysis of DNA from tail or ear biopsies (Table 1). Statistics. Statistical tests applied are indicated in the respective figure legends. MOG α α β β 2D2/TCR transgenic mice carry the V 3.2 J 18 and V 11DJ 1.1 regions Unpaired, two-tailed Student t test, two-way ANOVA, and two-tailed Pearson of the MOG-specific mouse T cell clone 2D2 (64). By virtue of their transgenic < + correlation test were performed. A P value 0.05 was considered statistically MOG-specific TCR expressed within the CD4 T cell compartment, 2D2/ significant. The asterisks depicted in the figures translate into the following MOG β TCR transgenic mice were genotyped via flow cytometry using V 11- groupings: *P < 0.05, **P < 0.01, ***P < 0.001. All quantitative analyses were specific antibodies and peripheral blood obtained from the tail vein. performed with Prism v5.0a for Mac OSX (GraphPad Software). Transgenic animals were identified due to the overrepresentation of this β-chain in their CD4+ T cell repertoire (64). Samples were acquired on a BD ACKNOWLEDGMENTS. We thank Dr. Noboru Mizushima (University of FACSCanto II using FACSDiva software v6.1.3 (BD Biosciences) and analyzed Tokyo) for providing the Atg5flox/flox mice, Dr. Fabienne Brilot (University with FlowJo software v9.3.1 (Tree Star). of Sydney) for providing the MOG-overexpressing and wild-type MO3.13 oligodendroglial cell lines, Anne Müller (University of Zurich) for excellent Induction of EAE. For induction of adoptively transferred EAE, C57BL/6 or 2D2/ technical assistance, Dr. Melanie Greter (University of Zurich) for valuable TCRMOG mice were used as donor mice. On day 0, donor mice were actively discussions, and the flow cytometry facility of the University of Zurich for induced with EAE via s.c. immunization in the flank region with 200 μg cell-sorting support. We thank Tara von Grebel of the Scientific Visualization and Visual Communication Department at the University of Zurich for her MOG – (MEVGWYRSPFSRVVHLYRNGK; RP10245; GenScript) in CFA (263810; 35 55 help with cartoon design. C.W.K. was supported by a scholarship provided by BD Difco). Pertussis toxin (200 ng) from Bordetella pertussis (179B; List Bi- the German Research Foundation (DFG Grant KE 1831/1-1) and a scholarship ological Laboratories) in PBS was administered i.p. After induction of EAE, mice from the University of Zurich (Forschungskredit FK-14-021). J.D.L. was sup- were observed daily for weight loss, disability, and availability of food and wa- ported by the Swiss National Foundation (31003A-169664), Novartis Foun- ter. On day 7 after immunization with MOG35–55/CFA, donor mice were eu- dation for medical-biological research, Sassella Foundation, Hartmann thanized with CO2. Spleen and draining lymph nodes were harvested and Müller Foundation, and Swiss Multiple Sclerosis Society.

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