Commensal Ro60 Orthologs as Persistent Triggers of Human Lupus

Background The earliest autoantibodies in systemic lupus erythematosus (SLE), a prototypical autoimmune disease, are directed against the RNA binding protein Ro60 but initiating triggers remain unknown. We identified commensal Ro60 orthologs in human skin, oral, and gut microbiomes in a subset of human species. Thus we hypothesize that Ro60‐ortholog‐carrying commensals induce autoimmunity via cross‐reactivity with human Ro60 in genetically susceptible individuals. Methods V4 16S rDNA samples from SLE and control subjects were sequenced using the MiSeq platform. In addition, species‐specific enrichment by real‐time PCR for Ro60 bacteria ( Propionibacterium propionicum, Corynebacterium amycolatum and Bacteroides thetaiotaomicron) was performed. Co‐immunoprecipitation was performed using human SLE serum and Ro60 ortholog‐containing bacterial lysates. Ro60‐specific memory CD4 T cells from SLE patients were cloned using a T cell library assay and stimulated with heat‐killed Ro60 bacteria. Supernatants were screened for cytokine proliferation using Legendplex. Ro60 −/− mice were crossed to the TLR7.1 C57BL/6 transgenic lupus model and screened for Ro60 antibodies. Germ‐free mice were monocolonized with B. thetaiotaomicron and tested for anti‐Ro60 antibodies by ELISA. Mesenteric lymph node (MLN) and spleen cells were stimulated with bacterial and human Ro60 to assess for proliferation in vitro. Results Ro60‐producing gut commensals were prevalent in controls and lupus patients. However, when human serum was used to co‐immunoprecipitate Ro60 and its bound Y RNA from the Ro60+ skin commensal P. propionicum , only antibodies from human Ro60‐positive lupus patients reacted with commensal Ro60. Lack of binding in Ro60‐negative patients or healthy controls supported antibody cross‐reactivity between human and commensal Ro60. Further, Human Ro60‐specific CD4 memory T cell clones proliferated in response to P. propionicum and commensal‐specific peptides , demonstrating T cell cross‐reactivity with commensal Ro60. Cytokine analysis proposes a heterogenous phenotype that is consistent with pathogen‐specific T cell clones. Serum anti‐Ro60 IgG autoantibodies were persistently induced in Ro60 −/− TLR7 tg mice, suggesting a microbial trigger in the absence of host Ro60. Finally, MLN and splenic lymphocytes from germ‐free C57BL/6 mice that were monocolonized with B. thetaiotaomicron proliferated in response to bacterial and human Ro60 and sera contained anti‐human Ro60 IgG antibodies. Conclusions Anti‐human Ro60 IgG in Ro60−/− TLR7 tg mice suggests a microbial trigger of these autoantibodies. Monocolonization with Ro60+ bacteria induced cross‐reactive anti‐human Ro60 responses in vivo. Ortholog cross‐reactivity is underscored by Ro60‐specific T cell clones and sera from lupus patients that reacted with commensal Ro60 in vitro. Our data support a model in which colonization with Ro60 ortholog‐carrying skin and gut bacteria sustain chronic autoreactivity in lupus. Quantifying and targeting Ro60+ bacteria in SLE patients may lead to novel biomarkers and treatment approaches. Ortholog cross‐reactivity is a novel concept that could contribute to the pathogenesis of human autoimmune diseases more broadly. Support or Funding Information Arthritis Foundation