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Select autophagy genes maintain quiescence of tissue-resident macrophages and increase susceptibility to Listeria monocytogenes

Abstract

Innate and adaptive immune responses that prime myeloid cells, such as macrophages, protect against pathogens1,2. However, if left uncontrolled, these responses may lead to detrimental inflammation3. Macrophages, particularly those resident in tissues, must therefore remain quiescent between infections despite chronic stimulation by commensal microorganisms. The genes required for quiescence of tissue-resident macrophages are not well understood. Autophagy, an evolutionarily conserved cellular process by which cytoplasmic contents are targeted for lysosomal digestion, has homeostatic functions including maintenance of protein and organelle integrity and regulation of metabolism4. Recent research has shown that degradative autophagy, as well as various combinations of autophagy genes, regulate immunity and inflammation5,6,7,8,9,10,11,12. Here, we delineate a function of the autophagy proteins Beclin 1 and FIP200—but not of other essential autophagy components ATG5, ATG16L1 or ATG7—in mediating quiescence of tissue-resident macrophages by limiting the effects of systemic interferon-γ. The perturbation of quiescence in mice that lack Beclin 1 or FIP200 in myeloid cells results in spontaneous immune activation and resistance to Listeria monocytogenes infection. While antibiotic-treated wild-type mice display diminished macrophage responses to inflammatory stimuli, this is not observed in mice that lack Beclin 1 in myeloid cells, establishing the dominance of this gene over effects of the bacterial microbiota. Thus, select autophagy genes, but not all genes essential for degradative autophagy, have a key function in maintaining immune quiescence of tissue-resident macrophages, resulting in genetically programmed susceptibility to bacterial infection.

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Fig. 1: Mice with deficiencies of certain autophagy genes in myeloid cells display enhanced resistance to L. monocytogenes.
Fig. 2: Alterations of peritoneal tissue-resident macrophages in mice with select autophagy gene deficiency.
Fig. 3: Disrupted immune cell homeostasis associated with Becn1 myeloid deficiency.
Fig. 4: IFN-γ signalling is necessary for macrophage activation and has a dominant effect over antibiotic-mediated immune quiescence in Becn1 deficiency.

Data availability

The data that support the findings of this study are available from the corresponding authors on reasonable request. RNA-seq data are available at the European Nucleotide Archive (PRJEB29191). Single-cell RNA-seq data are available at the GEO database (GSE121521).

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Acknowledgements

We thank G. J. Randolph, B. T. Edelson, D. Bhattacharya and the former members of the Virgin laboratory for discussion; J. R. Brestoff and S. Piersma for reading the manuscript; C. Yokoyama and H. Deng for technical assistance; and staff at the McDonnell Genome Institute, Genome Technology Access Center, Flow Cytometry Core Facility, Molecular Microbiology Imaging Facility, Pulmonary Morphology Core, and Center for Human Immunology & Immunotherapy Programs at Washington University School of Medicine for technical support. The research was supported by NIH grant U19 AI109725 and the Crohn’s and Colitis Foundation grant no. 326556 (to H.W.V.), NIH grant U19 AI42784 (to H.W.V. and C.L.S) and NIH grant R01 AI132697 (to C.L.S.). Authors receive support from MES of Russia (project 2.3300.2017/4.6; to K.Z.); NIH K08 (A128043; to C.B.W.); Pediatric Infectious Diseases Society/St Jude Children’s Research Hospital fellowship in basic research (to A.O.); Young Investigators Grant for Probiotics Research from the Global Probiotics Council (to M.T.B.); and Burroughs Wellcome Fund Investigators in the Pathogenesis of Infectious Disease (to C.L.S.).

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Y.-T.W. designed the project, performed experiments, analysed the data and wrote the manuscript. H.W.V. supervised project design and edited the manuscript. C.L.S. assisted with project design and edited the manuscript. Q.L., S.L., W.T.S., L.D. and C.B.W. performed experiments. K.-W.K., D.R.B., R.C.O., A.O., S.P., D.K. and M.T.B. assisted with experiments or project design. C.D. and S.A.H. helped to design RNA-seq experiments and analyse the data. K.Z. and M.N.A. analysed RNA-seq and single-cell RNA-seq data. J.D.P. analysed the histology. All of the authors read and edited the manuscript.

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Correspondence to Ya-Ting Wang or Christina L. Stallings or Herbert W. Virgin.

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H.W.V. is a founder of Casma Therapeutics and PierianDx. The work reported here was not funded by either company.

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Extended data

Extended Data Fig. 1 Mice with deficiencies of certain autophagy genes in myeloids cells display enhanced resistance to L. monocytogenes.

a, L. monocytogenes CFU in spleen or liver 3 days after infection of mice harboring myeloid deficiency (myeΔ) in multiple autophagy genes (data pooled from 2 experiments, Atg7f/f, n=9; Atg7mye, n=13; Atg16l1f/f, n=8; Atg16l1mye, n=10; Atg14f/f, n=8; Atg14mye, n=11 mice; mean ± s.e.m.; P by 2-tailed t test). b, Western blot analysis of p62, LC3 and GAPDH in peritoneal macrophages from naïve mice (Representative of n≥3 replicates). c, Ex vivo phagocytosis activity of peritoneal macrophages at 0 hour (data pooled from 2 experiments, Becn1f/f, n=12; Becn1mye, n=11; mean ± SEM; ns=not significant by 2-tailed t test). Source data

Extended Data Fig. 2 Mice with Beclin 1 deletion in DCs or neutrophils do not display L. monocytogenes resistance or macrophage activation phenotype.

a, Survival of mice harboring Beclin 1 deletion in CD11c+ and MRP8+ cells vs littermate controls, after i.p. inoculation with 4~5x105 CFUs of L. monocytogenes (Data pooled from 3-4 experiments; not significantly different by Log-rank Mantel-Cox test). b, Flow cytometry of ICAM2+ macrophage subsets in peritoneum of adult naïve mice (Data represents 2 experiments, Becn1f/f, n=3 vs Becn1f/f-Mrp8-cre, n=3; Becn1f/f, n=5 vs Becn1f/f-CD11c-cre, n=4; mean ± SEM; not significant by 2way ANOVA Sidak’s multiple comparisons).

Extended Data Fig. 3 Alterations of peritoneal tissue resident macrophages in mice with central autophagy gene deficiency.

a, b, c, d, Quantification of number of total cells, total ICAM2+ macrophages, and numbers of MHC-IIhigh and MHC-IIlow fractions of ICAM2+ macrophages from peritoneum lavage of mice harboring myeloid deficiency (myeΔ) in multiple autophagy genes and total deficiency (Δ) of Rubicon. (Data pooled from ≥3 independent experiments: Fip200f/f, n=14; Fip200mye, n=16; Atg5f/f, n=14; Atg5mye, n=12; Atg7f/f, n=8; Atg7mye, n=8; Atg16l1f/f, n=18; Atg16l1mye, n=13; Rubicon WT, n=10; Rubicon KO, n=9; Atg14f/f, n=16; Atg14mye, n=15 mice; mean ± SEM; P, or Padj for multiple comparison with 2-tailed t test). e, Violin plot showing percent of MHC-IIhighICAM2+ macrophages in total peritoneal immune cells (Atg14f/f, n=42; Atg14mye, n=38; Becn1f/f, n=12; Becn1mye, n=16; Fip200f/f, n = 15; Fip200myeΔ, n = 15; mean ± SEM; Padj by Kruskal–Wallis Dunn’s multiple comparison test).

Extended Data Fig. 4 Beclin 1 deficiency augmented baseline macrophage IFN signaling.

a, Volcano plot shows genes upregulated in macrophages from Becn1mye mice on the left and downregulated on the right in RNA-seq data set (Becn1f/f, n=4; Becn1mye, n=4). b, Gene set enrichment analysis of Becn1 dependent signature. (The green curve represents the density of the genes identified in the RNAseq with Normalized Enrichment Score (NES), P value and False Discovery Rate (FDR) listed.) c, Transcript levels of the indicated genes in naïve peritoneal macrophages. (3 independent experiments, Becn1f/f, n=9; Becn1mye, n=8; mean ± SEM; P by 2-tailed t test.).

Extended Data Fig. 5 DNA damage response and cell proliferation of Beclin 1 deficient peritoneal macrophages.

a, The presence of DNA double-strand break were revealed by immunofluorescence for γ-H2AX (red) in peritoneal macrophages treated with Bleomycin for 6 hours or untreated p62 is stained in green and nuclei were labeled by DAPI (blue). Cells displaying ≥10 γ-H2AX foci were counted as positive. (Data represents 2 independent experiments; n=4, mean ±SEM; P by 2-tailed t test.). b, Flow cytometry of BrdU incorporation by WT and Beclin 1- deficient ICAM2+ macrophages. (2 independent experiments; Becn1f/f, n=6 vs. Becn1mye, n=8; mean ±SEM; not significant by 2-tailed t test.), c, WT and Beclin 1-deficient ICAM2+ macrophages were enumerated after IL-4c injections (3 independent experiments; Becn1f/f+PBS, n=11; Becn1mye+PBS, n=16; Becn1f/f+IL4c, n=11; Becn1mye+ IL4c, n=19), and analyzed for frequency of BrdU+ and RELMα level (2 independent experiments; Becn1f/f+PBS, n=6; Becn1mye+PBS, n=9; Becn1f/f+IL4c, n=11; Becn1mye∆ + IL4c, n = 11; mean ± SEM; Padj by Tukey’s multiple comparisons test.).

Extended Data Fig. 6 Peritoneal lymphocytes changes revealed by Single-cell RNA sequencing.

a and c, Violin plots showing the expression of marker genes of B (a) and T (c) cells clusters by single cell RNAseq. Bar graph comparing fraction size of clusters. b and d Flow cytometry validation on naïve mice (Becn1f/f, n=9; Becn1myeΔ, n = 10; mean ± SEM; P and Padj for multiple comparison, by 2-tailed t test). e, Ifng transcript level among clusters revealed by single cell RNAseq.

Extended Data Fig. 7 Peritoneal macrophage activation in Becn1mye mice is independent of inflammasome and adaptive immune response.

a and b, Peritoneal macrophages obtained from naïve mice were analyzed for total cells (a), total ICAM2+ macrophages, MHC-IIhigh and MHC-IIlow fractions of ICAM2+ macrophages (b) by flow cytometry (Becn1f/fCasp1/11, n=6; Becn1myeCasp1/11, n=6 ; Becn1f/fRag1, n=7; Becn1mye∆Rag1, n = 8 mice, mean ± SEM, P and Padj by unpaired 2-tailed t test). c and d, Survival of mice after i.p. inoculation of 5x105 CFUs (c) or 5x104 CFU (d) of L. monocytogenes (Data pooled from 3-4 experiments, P by Log-rank Mantel-Cox test).

Extended Data Fig. 8 Ifngr rescues peritoneal immune cell homeostasis in Becn1mye mice.

ae, Flow cytometry analysis of total cells (a), B cells (b), T cells (c), SPM and monocytes (d), and peritoneal neutrophils (e) and obtained from naïve mice of the indicated genotypes. (Data are from 2 independent experiments; Becn1f/f, n=8 vs. Becn1mye, n=9; mean ± SEM; not significant by 2-tailed Mann-Whitney test.). f, Blood neutrophils were analyzed by flow cytometry. (n=6; mean ± SEM; P by 2-tailed Mann-Whitney test.).

Extended Data Fig. 9 Peritoneal macrophage activation in Becn1mye mice is independent of the presence of microbiota.

a and c, Quantification of total peritoneal cells and ICAM2+ macrophages (a) and numbers of ICAM2- macrophages and CD226- fraction of ICAM2- macrophages (c) (Padj by Dunn’s multiple comparisons test). Becn1f/f(Kool-Aid), n=7; Becn1f/f(abx), n=9; Becn1mye(Kool-Aid), n=9; Becn1mye(abx), n = 10; mean ± SEM, Padj by one-way ANOVA with Dunn’s multiple comparisons test). b, Quantification of 16S copy number from stool samples of mice. (Becn1f/f, n=7 each for kool-aid and abx vs. Becn1mye, n=5 each for kool-aid and abx; mean ± SEM, Padj analyzed by Tukey’s multiple comparisons test).

Extended Data Fig. 10 Becn1mye mice did not exhibit enhanced resistance to pulmonary influenza infection.

Mice were infected intranasally with 250 TCID50 influenza A PR8 and monitored for weight loss. (Data pooled from 4 independent experiments, mean ± SEM, not significant by 2way ANOVO for the whole curve or by 2-tailed t test for each time point).

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Wang, YT., Zaitsev, K., Lu, Q. et al. Select autophagy genes maintain quiescence of tissue-resident macrophages and increase susceptibility to Listeria monocytogenes. Nat Microbiol 5, 272–281 (2020). https://doi.org/10.1038/s41564-019-0633-0

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