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Research Article
AUTOIMMUNITY

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

Science Translational Medicine
28 Mar 2018
Vol 10, Issue 434

Autoimmune initiation by bacterial antigens

Lupus patients react to many self-proteins throughout the course of disease, with some of the earliest autoantibodies targeting the RNA binding protein Ro60. Greiling and colleagues sampled the microbiota of lupus patients and detected commensals with orthologs of human Ro60. These bacterial Ro60 proteins could be recognized by patient sera and stimulated patient T cells. Colonization of germ-free mice also led to human Ro60 reactivity and lupus-like symptoms, strongly indicating that molecular mimicry of the commensal Ro60 could be triggering autoreactivity and driving disease progression. These striking results have implications beyond lupus and could help uncover global mechanisms of autoimmune pathogenesis.

Abstract

The earliest autoantibodies in lupus are directed against the RNA binding autoantigen Ro60, but the triggers against this evolutionarily conserved antigen remain elusive. We identified Ro60 orthologs in a subset of human skin, oral, and gut commensal bacterial species and confirmed the presence of these orthologs in patients with lupus and healthy controls. Thus, we hypothesized that commensal Ro60 orthologs may trigger autoimmunity via cross-reactivity in genetically susceptible individuals. Sera from human anti-Ro60–positive lupus patients immunoprecipitated commensal Ro60 ribonucleoproteins. Human Ro60 autoantigen–specific CD4 memory T cell clones from lupus patients were activated by skin and mucosal Ro60-containing bacteria, supporting T cell cross-reactivity in humans. Further, germ-free mice spontaneously initiated anti-human Ro60 T and B cell responses and developed glomerular immune complex deposits after monocolonization with a Ro60 ortholog–containing gut commensal, linking anti-Ro60 commensal responses in vivo with the production of human Ro60 autoantibodies and signs of autoimmunity. Together, these data support that colonization with autoantigen ortholog-producing commensal species may initiate and sustain chronic autoimmunity in genetically predisposed individuals. The concept of commensal ortholog cross-reactivity may apply more broadly to autoimmune diseases and lead to novel treatment approaches aimed at defined commensal species.

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Supplementary Material

Summary

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.
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Science Translational Medicine
Volume 10 | Issue 434
March 2018

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Received: 14 March 2017
Accepted: 19 January 2018

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Acknowledgments

We thank all patients who have participated in this study as well as K. DeFrancesco and I. Matos for patient recruitment at the Yale Center for Clinical Investigation. We also thank J. Sterpka for technical assistance, L. Wen for providing GF NOD mice, N. Palm for use of an anaerobic chamber, M. Bosenberg for the dermatopathology samples, and S. Lewis for efforts to produce recombinant Ro60 protein. We thank W. Kwok (Benaroya Research Institute) for tetramer synthesis. Funding: This work was supported in part by grants from the NIH (K08AI095318, R01AI118855, R01 GM073863; T32AI07019), the Yale Rheumatic Diseases Research Core (NIH P30 AR053495), the Women’s Health Research at Yale, the O’Brien Center at Yale (NIH P30DK079310), the Arthritis National Research Foundation, the Arthritis Foundation, and the Lupus Research Institute. T.M.G. was supported by NIH training grant 5T32AR007016-41. K.H. was supported by NIH training grant T32GM007223 and by an NSF Predoctoral Fellowship. M.B. was supported by a Ruth L. Kirschstein National Research Service Award (F32 ES026227). The Yale Center for Clinical Investigation is supported by the Clinical and Translational Science Awards grant number UL1 RR024139 from the National Center for Research Resources, the National Center for Advancing Translational Science, and the NIH Roadmap for Medical Research. This work was also partly supported by the Intramural Research Program of the NIH, National Cancer Institute, Center for Cancer Research (Xinguo Chen, K.H., M.B., S.S., and S.L.W.) and by the NIH PREP program R25GM104553 (A.J.I.). Author contributions: S.S., P.D., and S.L.W. created the list of Ro60 orthologs; T.M.G. cross-referenced them with the commensal databases and created the phylogenetic tree; and S.S. identified YrlA RNAs. T.M.G., C.D., and J.G. led human subjects protocols and recruitment, and C.D., S.C.R., and C.K. assisted in human sample processing. K.H.C. provided sera and clinical information of Harvard cohort. T.M.G. and W.E.R. performed 16S sequencing and analysis. Anaerobic bacterial cultures were done by T.M.G. and C.D. T.M.G., S.C.R., and C.K. performed qPCR. Xinguo Chen, K.H., and M.B. expressed and purified recombinant bacterial Ro60 proteins. Xinguo Chen expressed and purified the human protein from baculovirus-infected cells with assistance from T.M.G. C.D. generated the hRo60 protein produced in mammalian cells. ELISA was designed by T.M.G. and C.D. and also performed by C.K. and Xindi Chen. T cell cloning, single-cell sorting, tetramer studies, T cell proliferation, and TCR sequencing were done by C.D. Human immunoprecipitation was done by M.B. Bacterial immunoprecipitation and Northern blots were done by Xinguo Chen with assistance from T.M.G. Western blotting was performed by C.D. 16S rRNA FISH experiments were performed by C.D. Immunofluorescence was performed by C.D. and A.J.I. Mouse experiments were performed by T.M.G. with help from S.M.V., W.E.R., and C.K. IMQ mouse experiments were performed by A.J.I. and D.Z.R. M.G. provided human subjects recruitment and experimental design. A.L.G. assisted in experiment design and critical review of the manuscript. M.A.K., T.M.G., C.D., and S.L.W. wrote the manuscript. M.A.K. and S.L.W. conceived the study and provided experiment design and guidance. Competing interests: The authors declare that they have no competing interests. Data and materials availability: Human sera of lupus patients from Massachusetts are available from Yale University under a material transfer agreement with Brigham and Women’s Hospital. The 16S sequencing data have been deposited in the European Nucleotide Archive, accession PRJEB24742.

Authors

Affiliations

Department of Immunobiology, Yale University School of Medicine, New Haven, CT 06510, USA.
Department of Dermatology, Yale University School of Medicine, New Haven, CT 06510, USA.
Present address: Department of Dermatology, Oregon Health and Science University, Portland, OR 97239, USA.
Department of Immunobiology, Yale University School of Medicine, New Haven, CT 06510, USA.
Department of Cell Biology, Yale University School of Medicine, New Haven, CT 06510, USA.
RNA Biology Laboratory, Center for Cancer Research, National Cancer Institute, Frederick, MD 21702, USA.
Present address: RNA Biology Laboratory, Center for Cancer Research, National Cancer Institute, Frederick, MD 21702, USA.
Department of Cell Biology, Yale University School of Medicine, New Haven, CT 06510, USA.
RNA Biology Laboratory, Center for Cancer Research, National Cancer Institute, Frederick, MD 21702, USA.
Present address: RNA Biology Laboratory, Center for Cancer Research, National Cancer Institute, Frederick, MD 21702, USA.
Department of Immunobiology, Yale University School of Medicine, New Haven, CT 06510, USA.
Department of Cell Biology, Yale University School of Medicine, New Haven, CT 06510, USA.
RNA Biology Laboratory, Center for Cancer Research, National Cancer Institute, Frederick, MD 21702, USA.
Present address: RNA Biology Laboratory, Center for Cancer Research, National Cancer Institute, Frederick, MD 21702, USA.
Department of Immunobiology, Yale University School of Medicine, New Haven, CT 06510, USA.
Department of Immunobiology, Yale University School of Medicine, New Haven, CT 06510, USA.
Department of Immunobiology, Yale University School of Medicine, New Haven, CT 06510, USA.
Department of Immunobiology, Yale University School of Medicine, New Haven, CT 06510, USA.
Department of Cell Biology, Yale University School of Medicine, New Haven, CT 06510, USA.
RNA Biology Laboratory, Center for Cancer Research, National Cancer Institute, Frederick, MD 21702, USA.
Present address: RNA Biology Laboratory, Center for Cancer Research, National Cancer Institute, Frederick, MD 21702, USA.
Christina Kriegel
Department of Immunobiology, Yale University School of Medicine, New Haven, CT 06510, USA.
Department of Immunobiology, Yale University School of Medicine, New Haven, CT 06510, USA.
Department of Immunobiology, Yale University School of Medicine, New Haven, CT 06510, USA.
Michael Girardi
Department of Dermatology, Yale University School of Medicine, New Haven, CT 06510, USA.
Patrick Degnan
Department of Microbial Pathogenesis and Yale Microbial Sciences Institute, Yale University School of Medicine, New Haven, CT 06510, USA.
Division of Rheumatology, Immunology and Allergy, Brigham and Women’s Hospital and Harvard Medical School, Boston, MA 02115, USA.
Department of Microbial Pathogenesis and Yale Microbial Sciences Institute, Yale University School of Medicine, New Haven, CT 06510, USA.
Department of Cell Biology, Yale University School of Medicine, New Haven, CT 06510, USA.
RNA Biology Laboratory, Center for Cancer Research, National Cancer Institute, Frederick, MD 21702, USA.
Present address: RNA Biology Laboratory, Center for Cancer Research, National Cancer Institute, Frederick, MD 21702, USA.
Department of Immunobiology, Yale University School of Medicine, New Haven, CT 06510, USA.
Section of Rheumatology, Department of Medicine, Yale University School of Medicine, New Haven, CT 06510, USA.

Funding Information

Ruth L. Kirschstein National Research Service Award: F32 ES026227
Women’s Health Research at Yale

Notes

*
These authors contributed equally as first authors.
§
Corresponding author. Email: [email protected] (M.A.K.); [email protected] (S.L.W.)

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