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Additional file 3: Supplementary Fig. 3. Memory function in 4 months old SPF, GF and ABX-treated 5xFAD mice. (A) Ratio of right versus left arm entries in the T-maze spontaneous alternation test of 10 months old SPF and GF or (B) SPF and ABX-treated 5xFAD and age-matched WT mice. (C-F) T-maze of 4 months old SPF and GF mice or (G-J) SPF and ABX-treated 5xFAD, as well as respective age-matched WT mice. (K-M) Novel object recognition task of 4 months old SPF and GF 5xFAD, as well as age-matched WT mice or (N-P) SPF and ABX-treated 5xFAD, as well as WT mice. Each symbol represents one mouse. Data are presented as mean ± s.e.m. Significant differences were determined by two-way ANOVA followed by Bonferroni’s post-hoc comparison test (*P

Additional file 1: Supplementary Fig. 1: Determination of intestinal bacterial loads and microbiota composition analysis. (A) Photograph of caeca from 4- and 10- months old SPF, GF and ABX-treated mice with ruler for scaling and (B) relative caecal weight referred to the body weight, and (C) absolute body weight. Each symbol represents one mouse. Data are presented as mean ± s.e.m. Significant differences were determined by one-way ANOVA followed by Tukey’s post-hoc comparison test (***P

Additional file 2: Supplementary Fig. 2. Absence of host microbiota reduces hippocampal Aβ depositions in 5xFAD mice. (A) Representative immunofluorescence images of 6E10+ (red) compact and diffuse Aβ plaques in the hippocampus of 4 months old SPF, GF and ABX-treated 5xFAD mice. Nuclei were stained with DAPI (blue). Overview of hippocampus and magnification of subiculum (dashed line) are shown. Scale bars represent 300 μm (overview) and 50 μm (insert). (B) Quantification of the number of 6E10+ Aβ-plaques per mm2, (C) percentage of 6E10+ area and (D) average 6E10+ plaque size (μm2) in coronal hippocampal sections of SPF, GF and ABX-treated 5xFAD mice. Each symbol represents one mouse. Data are presented as mean ± s.e.m. Significant differences were determined by one-way ANOVA followed by Tukey’s post-hoc comparison test (***P

Additional file 5: Supplementary Fig. 5. Gating of hippocampal microglia in 5xFAD mice and non-transgenic controls. Gating of CD11b+CD45low microglia from (A) 4 months old or (B) 10 months old SPF, GF and ABX-treated 5xFAD mice and age-matched WT controls. Representative dot plots are shown.

Additional file 4: Supplementary Fig. 4. Microglial density in hippocampus of 4 months old 5xFAD mice. (A) Representative immunofluorescence images of Iba1+ (green) microglia on coronal hippocampal sections of 4 months old SPF, GF and ABX-treated WT mice. Nuclei were stained with DAPI (blue). Scale bar: 50 μm. (B) Representative immunofluorescence images of 6E10 (red) and Iba1 (green) on coronal hippocampal sections of 4 months old 5xFAD mice. Nuclei were stained with DAPI (blue). Scale bar: 50 μm. (C) Quantification of 6E10+ plaque-associated microglia. Each symbol represents one mouse. Data are represented as means ± s.e.m. Significant differences were determined by one-way ANOVA followed by Tukey’s post-hoc comparison test (**P

Whereas central nervous system (CNS) homeostasis is highly dependent on tissue surveillance by immune cells, dysregulated entry of leukocytes during autoimmune neuroinflammation causes severe immunopathology and neurological deficits. To invade the CNS parenchyma, encephalitogenic T helper (TH) cells must encounter their cognate antigen(s) presented by local major histocompatibility complex (MHC) class II-expressing antigen-presenting cells (APCs). The precise mechanisms by which CNS-associated APCs facilitate autoimmune T cell reactivation remain largely unknown. We previously showed that mice with conditional deletion of the gene encoding the essential autophagy protein ATG5 in dendritic cells (DCs) are resistant to EAE development. Here, we report that the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 2, also known as CYBB/NOX2, in conventional DCs (cDCs) regulates endocytosed MOG (myelin oligodendrocyte protein) antigen processing and supports MOG-antigen presentation to CD4+ T cells through LC3-associated phagocytosis (LAP). Genetic ablation of Cybb in cDCs is sufficient to restrain encephalitogenic TH cell recruitment into the CNS and to ameliorate clinical disease development upon the adoptive transfer of MOG-specific CD4+ T cells. These data indicate that CYBB-regulated MOG-antigen processing and LAP in cDCs licenses encephalitogenic TH cells to initiate and sustain autoimmune neuroinflammation. Abbreviations: Ag: antigen; APC: antigen-presenting cell; AT: adoptive transfer; ATG/Atg: autophagy-related; BAMs: border-associated macrophages; BMDC: bone marrow-derived DC; CD: cluster of differentiation; CNS: central nervous system; CSF2/GM-CSF: colony stimulating factor 2 (granulocyte-macrophage); CYBB/NOX2/gp91phox: cytochrome b-245, beta polypeptide; DC: dendritic cell; EAE: experimental autoimmune encephalomyelitis; fl: floxed; FOXP3: forkhead box P3; GFP: green fluorescent protein; H2-Ab: histocompatibility 2, class II antigen A, beta 1; IFN: interferon; IL: interleukin; ITGAX/CD11c: integrin subunit alpha X; LAP: LC3-associated phagocytosis; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MFI: median fluorescence intensity; MG: microglia; MHCII: major histocompatibility complex class II; MOG: myelin oligodendrocyte glycoprotein; MS: multiple sclerosis; NADPH: nicotinamide adenine dinucleotide phosphate; ODC: oligodendroglial cell; OVA: ovalbumin; pDC: plasmacytoid DC; Ptd-L-Ser: phosphatidylserine; PTPRC: protein tyrosine phosphatase, receptor type, C; ROS: reactive oxygen species; SLE: systemic lupus erythematosus; TH cells: T helper cells; TLR: toll-like receptor; ZBTB46: zinc finger and BTB domain containing 46

Whereas central nervous system (CNS) homeostasis is highly dependent on tissue surveillance by immune cells, dysregulated entry of leukocytes during autoimmune neuroinflammation causes severe immunopathology and neurological deficits. To invade the CNS parenchyma, encephalitogenic T helper (TH) cells must encounter their cognate antigen(s) presented by local major histocompatibility complex (MHC) class II-expressing antigen-presenting cells (APCs). The precise mechanisms by which CNS-associated APCs facilitate autoimmune T cell reactivation remain largely unknown. We previously showed that mice with conditional deletion of the gene encoding the essential autophagy protein ATG5 in dendritic cells (DCs) are resistant to EAE development. Here, we report that the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 2, also known as CYBB/NOX2, in conventional DCs (cDCs) regulates endocytosed MOG (myelin oligodendrocyte protein) antigen processing and supports MOG-antigen presentation to CD4+ T cells through LC3-associated phagocytosis (LAP). Genetic ablation of Cybb in cDCs is sufficient to restrain encephalitogenic TH cell recruitment into the CNS and to ameliorate clinical disease development upon the adoptive transfer of MOG-specific CD4+ T cells. These data indicate that CYBB-regulated MOG-antigen processing and LAP in cDCs licenses encephalitogenic TH cells to initiate and sustain autoimmune neuroinflammation. Abbreviations: Ag: antigen; APC: antigen-presenting cell; AT: adoptive transfer; ATG/Atg: autophagy-related; BAMs: border-associated macrophages; BMDC: bone marrow-derived DC; CD: cluster of differentiation; CNS: central nervous system; CSF2/GM-CSF: colony stimulating factor 2 (granulocyte-macrophage); CYBB/NOX2/gp91phox: cytochrome b-245, beta polypeptide; DC: dendritic cell; EAE: experimental autoimmune encephalomyelitis; fl: floxed; FOXP3: forkhead box P3; GFP: green fluorescent protein; H2-Ab: histocompatibility 2, class II antigen A, beta 1; IFN: interferon; IL: interleukin; ITGAX/CD11c: integrin subunit alpha X; LAP: LC3-associated phagocytosis; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MFI: median fluorescence intensity; MG: microglia; MHCII: major histocompatibility complex class II; MOG: myelin oligodendrocyte glycoprotein; MS: multiple sclerosis; NADPH: nicotinamide adenine dinucleotide phosphate; ODC: oligodendroglial cell; OVA: ovalbumin; pDC: plasmacytoid DC; Ptd-L-Ser: phosphatidylserine; PTPRC: protein tyrosine phosphatase, receptor type, C; ROS: reactive oxygen species; SLE: systemic lupus erythematosus; TH cells: T helper cells; TLR: toll-like receptor; ZBTB46: zinc finger and BTB domain containing 46