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USP15 regulates type I interferon response and is required for pathogenesis of neuroinflammation

An Erratum to this article was published on 16 November 2016

This article has been updated

Abstract

Genes and pathways in which inactivation dampens tissue inflammation present new opportunities for understanding the pathogenesis of common human inflammatory diseases, including inflammatory bowel disease, rheumatoid arthritis and multiple sclerosis. We identified a mutation in the gene encoding the deubiquitination enzyme USP15 (Usp15L749R) that protected mice against both experimental cerebral malaria (ECM) induced by Plasmodium berghei and experimental autoimmune encephalomyelitis (EAE). Combining immunophenotyping and RNA sequencing in brain (ECM) and spinal cord (EAE) revealed that Usp15L749R-associated resistance to neuroinflammation was linked to dampened type I interferon responses in situ. In hematopoietic cells and in resident brain cells, USP15 was coexpressed with, and functionally acted together with the E3 ubiquitin ligase TRIM25 to positively regulate type I interferon responses and to promote pathogenesis during neuroinflammation. The USP15-TRIM25 dyad might be a potential target for intervention in acute or chronic states of neuroinflammation.

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Figure 1: An ENU-induced mutation in Usp15 protects mice against development of ECM.
Figure 2: Reduced protein expression and reduced stability of the USP15 L749 variant in vivo and in vitro.
Figure 3: Diminished cerebral pathogenesis of ECM and EAE in homozygous Usp15L749R mice.
Figure 4: Effect of USP15 on global gene expression during neuroinflammation of the brain and of the spinal cord.
Figure 5: Cell populations and associated molecular pathways regulated differentially in a USP15-dependent fashion.
Figure 6: USP15 modulates the type I interferon response through interaction with TRIM25.
Figure 7: Cellular compartments responsible for USP15-dependent effects in neuroinflammation and in type I interferon responses.

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Change history

  • 24 October 2016

    In the version of this article initially published online, the symbol for the gene encoding granzyme B was incorrect (Gmzb) in the text in the third paragraph of the fourth subsection of Results and Figure 5d, and the symbol for the gene encoding granzyme A was incorrect (Gmza) in Figure 6h. These should be Gzmb and Gzma, respectively. The errors have been corrected for the print, PDF and HTML versions of this article.

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Acknowledgements

We thank R. Van Bruggen, S. Gauthier, P. D'Arcy and G. Perreault for assistance in the ENU project, and C. Meunier for technical assistance. Supported by the Canadian Institutes of Health Research (MOP119342 and MOP133487 to P.G. and S.V.) and Amorchem (PT63088).

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Authors

Contributions

S.T., M.L., J.M. and J.B. contributed to mutation identification. S.T. performed all of the PbA experiments. M.J.P. performed all of the EAE experiments. S.T. and M.J.P. performed biochemical work. I.R., J.M.K. and S.T. performed the immunophenotyping experiments. D.L. performed RNA sequencing analyses, and S.T. carried out validations by RT-qPCR. Primary cells from brain were provided and characterized by J.A., N.A., A.P., M.J.P. and L.M.H., with additional contribution from G.A.L.-T., S.I., K.M., C.L. and K.P.K. kindly provided gene knockout animals. S.T. performed Listeria experiments with guidance from C.M.K. N.F. performed BM transplant experiments. J.B., J.M. and M.L. performed analyses of exome sequences. P.G. and S.M.V. supervised the project, helped to design experiments and analyzed data. P.G., S.T., D.L., M.J.P. and N.F. wrote the first draft of the manuscript. All of the authors provided helpful comments on the manuscript.

Corresponding author

Correspondence to Philippe Gros.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Ubiquitous pattern of Usp15 mRNA expression in embryonic, post-natal and adult mice

Mouse sections were stained with cresyl violet to localize Usp15 RNA to specific organs and structures. In situ hybridization was carried out using radiolabelled antisense (as) and sense (s) probes. The results shown are from X-ray film autoradiography obtained following 5-days exposure. Non-specific localized signals (visible with sense and anti-sense probes) are indicated with an asterisk (*); in the teeth (p10) and the large intestine lumen (p10 and adult). (Magnification: Embryonic x2.4, Post-natal x3, Adult x2.4). Abbreviations: Adr–adrenal gland; At–heart atrium; Br–brain; Bro–bronchcus; Car– cartilage; Cb–cerebellum; Co–colon; Cx–cerebral cortex; Du – duodenum; E – eye; Ep – epididymis; Es – esophagus; GB – gallbladder; HV–heart ventricle; Il–ileum; Je–jejunum; Ki–kidney; Li–liver; LI–large intestine; Lu–lung; OL–olfactory lobe; Ov–ovary; Ovi–oviducts; PB–pelvis bone; Pc–pancreas; PG–pituitary gland; Pr–prostate; PTh–parathyroid gland; R–ribs; Sk–skin; Spl–spleen; St–stomach; SV–seminal vesicle; Te–testis; Th–thyroid gland; UB–urinary bladder; Ut-uterus; CA–central artery; GC-germinal center; LN–lymphatic nodule; RP–red pulp; Tr–trabeculum; V-vein; LF–lymphoid follicle; Me–medulla; MG–mammary glands; Cx–cortex.

Supplementary Figure 2 Reduced protein stability of the USP15 L720R human variant in vitro

HEK293 cells stably expressing HA-tagged WT or USP15L720R proteins were treated with cycloheximide (CHX, 20 μg/ml) for 2, 4, 8, and 16h, and equal amounts of protein were analyzed by immunoblotting. Data is from a single experiment.

Supplementary Figure 3 Immunophenotyping of Usp15L749R mutants at steady-state and following P. berghei ANKA infection

(a) The number and proportions of different spleen cell types from naïve and from day 5-PbA infected animals, were established by flow cytometry with markers for T cells (CD4, CD8), B cells (B220), NK cells (NK1.1), monocytes and neutrophils (CD11b, Ly6G). Results are pooled from 5 independent experiments. (b) The percentage of splenic CD4+ and CD8+ effector T cells (CD62LCD44+) were also assessed. Data represents a single experiment with 5 mice per group, and are expressed as a mean ± SD. (c-d) Cells were re-stimulated in vitro with either media alone (unstimulated, US), with anti-CD3 and anti-CD28 (TCR engagement), with PMA/Ionomycin, with CpG, or with Poly:IC and cytokine production was assessed by flow cytometry (C, intracellular staining), or by ELISA (D, culture supernatants). Data is a representation of two independent experiments with 5 mice per group, and is expressed as a mean ± SD. (e) The activation state of CD4+ and CD8+ T cells were assessed by analysis of CD69 cell surface expression in response to TCR engagement (anti-CD3/anti-CD28). Data represents a single experiment with 5 mice per group, and is expressed as a mean ± SD.

Supplementary Figure 4 USP15 negatively regulates CD4+ T cell activation during Listeria monocytogenes infection

Wild type B6 mice and Usp15L749R mutants infected with 1x104 CFU of Listeria monocytogenes (strain 10403s) expressing ovalbumin (OVA) were sacrificed on day 7 post-infection, and phenotyped for the activation of the T cell response in spleen cell populations. (a, b) CD44 expression (T cell activation) on CD4+ T cells (A), or CD8+ T cells (B), expressed as percentage and total cell numbers. (c, d) Cells were re-stimulated in vitro with Listeria-specific antigens, LLO or OVA, and IFN-γ production was assessed by flow cytometry (C, intracellular staining), or by ELISA (D, culture supernatants) for CD4+ and CD8+ T cells. (e) Serum IFN-γ levels were measured by ELISA, and plotted as optical density absorbance (OD) at 450 nm. (a-e) Data is a combination of two independent experiments. All data are expressed as a mean ± SD for each group, and all statistical analyses were performed using the two-tailed unpaired Student’s t-test.

Supplementary Figure 5 Cell populations and associated molecular pathways differentially regulated in a USP15-dependent fashion

(a) LEA dendogram for genes with reduced expression in Usp15L749R mutant mice compared to B6 (day 5 post-PbA infection) and that drive significant enrichment (FDR<0.01) of immunological expression signatures (GSEA). Enriched immunological signatures and functions are highlighted by color boxes: red = signatures of IFN activation, green = myeloid signatures and responses, and purple = T cell signatures. Refer to Online Methods for details on LEA analysis. (b) LEA clustering analysis as described in (A) for immunological signatures depleted in Usp15L749R mutant mice during EAE neuroinflammation progression.

Supplementary Figure 6 Mouse mutants bearing a loss of function mutation in Irf3 are protected against neuroinflammation

Survival plots for PbA-infected (a) Irf3 mutants (Irf3-/-) (n=13) and B6 controls (n=8), and (b) Mavs mutants (Mavs-/-) (n=22) and B6 (n=11). Statistical significance for survival between groups of mice was determined by the Log-rank test (* P<0.05, **** P<0.0001).

Supplementary Figure 7 The L720R mutation affects the ability of USP15 to deubiquitinate SMURF2

HEK293T cells transiently expressing Smurf2-FLAG with or without Usp15-Xpress construct plus HA-tagged ubiquitin (Ub) were lysed 48h post-transfection, SMURF2 was immunoprecipitated using anti-FLAG antibody and immunoblotting analysis was performed as indicated. Construct expression in whole cell lysates (WCL) was confirmed by western blot

Supplementary Figure 8 Full gating strategy for flow cytometry analysis of brain cellular infiltration

Five days post-PbA infection, infiltrating cells were isolated from perfused brains and stained for flow cytometry analyses. Viable cells were selected for based on their exclusion of the Zombie Aqua Dye. Leukocytes were gated as CD45hi cells. Populations of leukocytes gated from the CD45hi gate were analyzed as follows: CD4 T cells (TCRb+CD4+CD8), CD8 T cells (TCRb+CD4CD8+), macrophages (Cd11b+F4/80+) and neutrophils (Cd11b+Ly6G+).

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–8 (PDF 1128 kb)

Supplementary Table 1

List of dys-regulated genes in Usp15L749R mutant mice undergoing ECM and EAE models of neuroinflammatory diseases, related to Fig. 4b. (XLSX 120 kb)

Supplementary Table 2

Detailed matrix of leading edge analysis clustering performed on ECM and EAE depleted gene signatures in Usp15L749R mutant mice, related to Fig. 5a. (XLSX 462 kb)

Supplementary Table 3

qPCR validation primers, related to Fig. 5c-f. (XLSX 780 kb)

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Torre, S., Polyak, M., Langlais, D. et al. USP15 regulates type I interferon response and is required for pathogenesis of neuroinflammation. Nat Immunol 18, 54–63 (2017). https://doi.org/10.1038/ni.3581

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