Research ArticleAutoimmunity

Commensal orthologs of the human autoantigen Ro60 as triggers of autoimmunity in lupus

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Science Translational Medicine  28 Mar 2018:
Vol. 10, Issue 434, eaan2306
DOI: 10.1126/scitranslmed.aan2306
  • Fig. 1 The human commensal microbiota contains multiple species with Ro60 orthologs.

    (A) Phylogenetic tree showing commensal bacterial ortholog Ro60 sequence homology to hRo60, with commensal niches identified by colors of each branch (see legend). (B) Structure of hRo60 with a major B cell epitope [amino acids (aa) 169 to 190] mapped in pink and T cell epitope (amino acids 316 to 335) mapped in orange, and the protein sequence alignments between human and commensal Ro60 at these epitopes compared below. Green color indicates residues that interact with Y RNA. Blue color indicates residues that interact with misfolded RNA. P. prop, P. propionicum; C. amyc, C. amycolatum; A. mass, A. massiliensis.

  • Fig. 2 Ro60 commensal bacteria are common among lupus and healthy subjects without overt dysbiosis of the fecal, oral, or skin microbiome.

    (A) 16S V4 sequencing was performed from the fecal, oral, and skin microbiomes of 15 subjects with lupus and 7 healthy controls. Principal coordinates analysis of weighted UniFrac distances represents β-diversity by body site but not by the presence of serum anti-Ro60 antibodies. (B) Vertical bars represent relative abundance of microbial phyla in individual fecal, oral, and skin microbiome samples. Legend indicates most abundant phyla. No significant differences in linear discriminant analysis effect size were found between groups. (C) Heat map of relative abundance of four Ro60 commensal bacteria from human microbiome samples measured by bacterial Ro60-specific qPCR. Each row represents a study subject: healthy (NOR), SLE, or SCLE. (+) or (−) indicates serum anti-Ro60 immunoglobulin G (IgG) autoantibodies. Subjects completed up to three longitudinal visits, shown by column. White space indicates no sample. Legend indicates color relative to ΔΔCt value. There was no significant difference in the mean abundance between Ro60(+) and Ro60(−) individuals except P. prop from the chest (two-sample t test, *P = 0.02) (plot shown at the bottom right). (D) SCLE patient cutaneous lesional biopsies stained with a P. prop 16S rDNA–specific FISH probe (green) as well as the eubacterial probe EUB338 (red). DAPI, 4′,6-diamidino-2-phenylindole.

  • Fig. 3 Commensal-reactive T cell clones from lupus patients cross-react with hRo60 protein and a pathogenic Ro60 T cell peptide.

    Human memory T cells from an anti-Ro60–positive SLE patient were sorted into (A) CCR6 and (B) CCR6+ subsets and stimulated with the Ro60 commensal P. prop. X axis indicates SI, and y axis indicates proliferation as counts per minute (cpm). Each point on the graph represents one clone. Dotted line indicates autologous, irradiated monocyte control. (C) Restimulation of CCR6+ clones with SI ≥ 5 with recombinant hRo60 or the pathogenic Ro60 T cell peptide p316-335. (D) Identical experiment as in (A) to (C), stimulating CD4+ memory CCR6 and (E) CCR6+ lupus T cells from another anti-Ro60–positive SLE patient with recombinant bacterial Ro60 from the Ro60-containing gut commensal B. theta. (F) Similar to (C), CCR6+ B. theta Ro60 (BtRo60)–reactive T cell clones were restimulated with recombinant hRo60 or the pathogenic Ro60 T cell peptide p316-335.

  • Fig. 4 hRo60-reactive patient T cell clones generated by a CD4+ T cell library assay cross-react with the Ro60 commensal P. prop.

    Human lupus memory T cells were sorted into (A) CCR6 and (B) CCR6+ subsets and stimulated with hRo60. X axis indicates SI, and y axis indicates proliferation as counts per minute (cpm). Each point on the graph represents one clone. (C) Three clones from each subset had SI ≥ 5 and were restimulated with the Ro60 ortholog–containing commensal bacteria B. theta and P. prop (two-sample t test, *P < 0.05). (D) Cytokine concentrations (in picograms per milliliter) from the supernatant of the cross-reactive clone 72 hours after stimulation. In (C) and (D), measurements were performed in duplicates, as indicated by the dots in each group. Dotted line indicates monocyte control.

  • Fig. 5 hRo60-reactive T cell clones cross-react with commensal Ro60-reactive mimic peptides and whole commensal bacteria.

    (A) Alignment of hRo60 T cell autoepitope peptide 369–383 with the corresponding amino acid sequences in commensal Ro60 orthologs of P. prop., C. amyc, and B. theta. CCR6 (B) and CCR6+ (C) memory CD4+ T cell subsets of an anti-Ro60–positive SLE patient stimulated with hRo60 protein. Y axis indicates proliferation as reactive light units (RLU) using a nonradioactive adenosine 5′-triphosphate (ATP) release assay. Dotted lines indicate monocyte control, which was set as baseline to zero for restimulation assays. (D) hRo60-reactive T cell clones of the same patient proliferated to hRo60 peptide 369–383, commensal ortholog mimic peptides, and whole commensal bacteria. (E) CD4+ T cell clone isolated from the peripheral blood of an anti-Ro60–positive SLE patient using a hRo60 peptide–specific tetramer cross-reacts with the Ro60 commensal C. amyc (*P < 0.05, two-sample t test).

  • Fig. 6 Human lupus sera immunoprecipitate YrlA RNA–containing RNPs from P. prop lysates.

    (A) After incubating P. prop lysates with human sera, RNAs in immunoprecipitates were extracted and subjected to Northern blotting to detect YrlA. First lane, molecular size markers (nucleotides). Total P. prop, total RNA extracted from the input lysate. Serum from a healthy donor is shown in the next lane. Six SLE sera and two SCLE sera are labeled with + or − representing the anti-Ro60 IgG antibody status by ELISA. (B) Predicted structure of P. prop YrlA. (C) As a negative control, the blot was reprobed for P. prop tRNA-proline-GGG. (D) Lupus sera from independent Harvard patient cohort (listed by identification numbers).

  • Fig. 7 Anti-Ro60–positive SLE sera bind the recombinantly expressed BtRo60 ortholog by Western blot.

    (A) Sera from four Yale SLE patients with positive serologies for anti-hRo60 IgG were subjected to Western blotting with recombinant hRo60 (60kDa), recombinant BtRo60 (~56.5 kDa) and bacterial lysates from B. theta, as well as two skin/gut commensal strains that do not carry Ro60 orthologs, P. acnes and R. intestinalis (R. intes). (B) Sera from three Harvard SLE patients with positive serologies for anti-hRo60 IgG were subjected to Western blotting as in (A).

  • Fig. 8 Monocolonization of the GF mouse gut with B. theta leads to hRo60 T and B cell responses.

    (A) hRo60 ELISA of B. theta monocolonized C57Bl/6 mice (n = 15) after 3 to 5 months of colonization compared to GF age- and sex-matched controls of the C57Bl/6 strain (n = 2). X axis shows twofold serial dilutions of sera from 1:250 to 1:4000, with the mean and SD of duplicate measurements plotted for each mouse. (B) Proliferation, measured by an ATP release assay, of cells from MLNs and (C) spleens from B. theta–monocolonized C57Bl/6 mice, stimulated with B. theta lysate or recombinant BtRo60 protein for 72 hours in triplicate. Each point represents the SI (proliferation divided by the mean background proliferation from cells with no antigen) of MLNs and spleens pooled by cage. P values were calculated using two-sample t tests. (D) hRo60 ELISA of B. theta–monocolonized, autoimmune-prone NOD mice (n = 6) after 2 weeks of colonization, compared to two age-matched GF mice (6-week-old C57Bl/6 females). X axis shows twofold serial dilutions of sera from 1:250 to 1:4000, with the mean and SD of duplicate measurements plotted for each mouse. (E) Proliferation assay of cells from MLNs and spleens (F) from monocolonized NOD mice stimulated with B. theta lysate, BtRo60, hRo60, and control proteins (BSA and β2GP1, an unrelated autoantigen) for 72 hours in triplicate as above. (G to L) Eight-week-old GF C57Bl/6 mice were monocolonized with B. theta followed by topical treatment with or without a TLR7 agonist (IMQ) for 8 weeks. Monocolonized mice were compared to age- and sex-matched GF C57Bl/6 mice with and without IMQ (n = 3 to 7 in each of the four groups). (G and H) Comparison of body weight and relative spleen weight in B. theta–monocolonized mice with and without IMQ versus GF controls. (I to K) Anti-hRo60, anti-dsDNA, and anti-RNA IgG ELISAs of B. theta–monocolonized and GF mice with or without IMQ after 8 weeks of treatment. B. theta monocolonization together with IMQ. (L) Renal immunofluorescence of B. theta–monocolonized and GF mice treated with or without IMQ. Immunofluorescence of kidneys was performed on kidney tissue from B. theta–monocolonized mice with or without IMQ and GF with IMQ. Anti-C3 (green), anti-C1q (teal), and anti-IgG, anti-IgA, and anti-IgM (red). OD, optical density; N.S., not significant. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, two-sample t test.

  • Table 1 Human subject cohort and clinical characteristics.

    SCLE02, SCLE03, SCLE05, and SCLE06 were de-identified serum or biopsy samples, so additional clinical information was unavailable. Ro60 column indicates the presence or absence of anti-Ro60 serum IgG. F, female; M, male; HCQ, hydroxychloroquine; NA, not available. Additional clinical information is available in tables S2 and S3.

    SubjectDiagnosisSexAge (years)HLA-DR3HLA-DR15Ro60Immunosuppressive
    medications
    HCQ
    SLE01SLEF40+Azathioprine
    SLE02SLEF47++Rituximab
    Prednisone 5 mg daily
    SLE03SLEF49Mycophenolate mofetil
    Prednisone 7.5 mg daily
    +
    SLE04SLEF29+Discontinued
    mycophenolate mofetil
    and prednisone against
    medical advice 1 month
    prior
    SLE05SLEF49++Prednisone 1 mg daily+
    SLE06SLEF40+Mycophenolate mofetil
    Prednisone 5 mg daily
    +
    SLE07SLEF31+
    SLE08SLEF52++
    SLE09SLEF34++
    SLE10SLEF47++++
    SLE11SLEF31++Mycophenolic acid
    Prednisone 10 mg daily
    +
    SLE12DLEF54++
    SLE13SLEF26++Mycophenolic acid
    Methotrexate
    Prednisone 7.5 mg daily
    +
    SLE14SLEF32
    SLE15SLEF44+
    SLE18SLEF33+NANA
    SCLE01SCLEF75++Azathioprine+
    SCLE04SCLEM72+

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/10/434/eaan2306/DC1

    Materials and Methods

    Fig. S1. Sequence alignment of full-length hRo60 and selected commensal orthologs.

    Fig. S2. Coimmunoprecipitation of lupus study subject sera confirmed anti-Ro60 antibody status.

    Fig. S3. α-Diversity represented by box plots of the Shannon-Weiner diversity index.

    Fig. S4. SCLE skin eruption.

    Fig. S5. TT-reactive CD4+ T cell clone from a healthy donor generated by a CD4 T cell library assay.

    Fig. S6. Ro60-negative SLE patient CD4+ T cells lack reactivity to hRo60 protein.

    Fig. S7. Cytokine concentrations (pg/ml) of supernatants from the cross-reactive T cell clone from Fig. 5 measured using a bead-based immunoassay.

    Fig. S8. Anti-Ro60 antibody status of Harvard cohort lupus subjects.

    Fig. S9. YrlA RNA is not detected in immunoprecipitates from B. theta using human lupus sera.

    Fig. S10. B. theta monocolonization of GF mice induces gut and systemic immune changes.

    Fig. S11. Schematic of proposed mechanism of how Ro60 bacteria trigger and sustain autoimmunity.

    Table S1. Commensal bacterial Ro60 orthologs identified by in silico methods.

    Table S2. Lupus study subject clinical data.

    Table S3. Healthy control study subject clinical data.

    Table S4. Efficiency and specificity of bacterial Ro60 qPCR primers.

    Table S5. Primary data.

    References (6377)

  • Supplementary Material for:

    Commensal orthologs of the human autoantigen Ro60 as triggers of autoimmunity in lupus

    Teri M. Greiling, Carina Dehner, Xinguo Chen, Kevin Hughes, Alonso J. Iñiguez, Marco Boccitto, Daniel Zegarra Ruiz, Stephen C. Renfroe, Silvio M. Vieira, William E. Ruff, Soyeong Sim, Christina Kriegel, Julia Glanternik, Xindi Chen, Michael Girardi, Patrick Degnan, Karen H. Costenbader, Andrew L. Goodman, Sandra L. Wolin,* Martin A. Kriegel*

    *Corresponding author. Email: martin.kriegel{at}yale.edu (M.A.K.); sandra.wolin{at}nih.gov (S.L.W.)

    Published 28 March 2018, Sci. Transl. Med. 10, eaan2306 (2018)
    DOI: 10.1126/scitranslmed.aan2306

    This PDF file includes:

    • Materials and Methods
    • Fig. S1. Sequence alignment of full-length hRo60 and selected commensal orthologs.
    • Fig. S2. Coimmunoprecipitation of lupus study subject sera confirmed anti-Ro60 antibody status.
    • Fig. S3. α-Diversity represented by box plots of the Shannon-Weiner diversity index.
    • Fig. S4. SCLE skin eruption.
    • Fig. S5. TT-reactive CD4+ T cell clone from a healthy donor generated by a CD4 T cell library assay.
    • Fig. S6. Ro60-negative SLE patient CD4+ T cells lack reactivity to hRo60 protein.
    • Fig. S7. Cytokine concentrations (pg/ml) of supernatants from the cross-reactive T cell clone from Fig. 5 measured using a bead-based immunoassay.
    • Fig. S8. Anti-Ro60 antibody status of Harvard cohort lupus subjects.
    • Fig. S9. YrlA RNA is not detected in immunoprecipitates from B. theta using human lupus sera.
    • Fig. S10. B. theta monocolonization of GF mice induces gut and systemic immune changes.
    • Fig. S11. Schematic of proposed mechanism of how Ro60 bacteria trigger and sustain autoimmunity.
    • Table S1. Commensal bacterial Ro60 orthologs identified by in silico methods.
    • Table S2. Lupus study subject clinical data.
    • Table S3. Healthy control study subject clinical data.
    • Table S4. Efficiency and specificity of bacterial Ro60 qPCR primers.
    • References (6377)

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Table S5 (Microsoft Excel format). Primary data.

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