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Overview

Translational Research in Lyme Disease and Rheumatoid Arthritis

The Laboratory of Allen Steere, MD, and Klemen Strle, PhD, at Massachusetts General Hospital specializes in the identification of novel antigens, including those from infectious agents or self-proteins, the elucidation of immune responses to these antigens, and the determination of microbial and host genetic factors that underlie adverse clinical outcomes. This work requires a combination of discovery-based genomic, transcriptomic and proteomic approaches as well as functional studies using cells and affected tissues. These include determinations of T cell and antibody phenotypes and function and deep characterization of implicated immune cells using genomics and transcriptomics. This translational research, which is based on the study of human patients, has implications for pathogenesis, diagnosis and treatment of patients with these diseases.

Lyme Arthritis

Background
Although most patients with Lyme arthritis (LA) respond well to antibiotic therapy, some patients develop persistent synovitis after treatment lasting months to several years. This is called post-infectious, antibiotic-refractory LA. In these patients, synovial tissue, the target of the immune response, is similar with that seen in other forms of chronic inflammatory arthritis, including rheumatoid arthritis (RA).

We are studying post-infectious LA in which the initial triggering cause, Borrelia burgdorferi, is known with certainty as a human model to better understand how infection may trigger immune dysregulation and autoimmunity affecting joints in these diseases. In LA, risk factors for this untoward outcome include infection with certain highly inflammatory strains of B. burgdorferi, excessive joint inflammation characterized by high levels of IFNγ and inadequate IL-10, host genetic factors including a TLR1 polymorphism and certain HLA-DR alleles, and immune dysregulation of the CD4+eff/reg ratio, leading to pathogenic autoimmunity.

Discovery-based Approach for the Identification of Pathogenic Autoimmune Responses
The identification of relevant autoantigens has been a challenge in any autoimmune disease. However, new discovery-based approaches offer innovative approaches. In collaboration with Dr. Catherine Costello, who directs the Biomedical Mass Spectrometry Center at Boston University, we have developed a highly innovative, discovery-based approach for the identification of naturally presented HLA-DR peptides (T cell epitopes) in synovial tissue, synovial fluid mononuclear cells and peripheral blood mononuclear cells. With this method, HLA-DR-presented peptides are eluted and identified using tandem mass spectrometry and proteomic databases, and identified peptides are then tested for T and B cell reactivity using patient samples. In this way, four novel autoantigens have been identified, endothelial cell growth factor (ECGF), matrix metalloproteinase-10 (MMP-10), apolipoprotein B-100 and annexin A2, as targets of T and B cell responses in patients with antibiotic-refractory LA. Moreover, the antibody responses to these antigens correlate with Th17 immunity and specific aspects of synovial pathology.

Hypothesis to Explain Post-infectious, Antibiotic-refractory LA
In patients with post-infectious LA, transcriptomic analyses of synovial tissue reveal up-regulation of genes associated with IFN-γ responses, MHC class II antigen processing and presentation, and cell-mediated cytotoxicity. Our hypothesis is that especially high levels of IFN-γ, which are associated with certain genetic polymorphisms in immune cells, result in high expression of HLA-DR molecules not only on professional antigen presenting cells (APCs), but also on non-professional APCs, such as endothelial cells and fibroblast-like synoviocytes. Cytotoxic lymphocytes may be directed against these non-professional APCs, which are recognized as abnormal cells. The result is vascular damage, autoimmune or autoinflammatory processes, fibrosis and synovial hyperplasia lasting months to years, even in the absence of active infection.

Rheumatoid Arthritis

Background
Great progress has been made in the identification of genetic factors and inflammatory disease pathways that influence rheumatoid arthritis (RA), but environmental factors are only now being determined. A key hypothesis is that specific organisms in the gut, lung or oral microbiota may shape mucosal and systemic responses that affect joints in RA patients.

In RA, we are studying the possible role of several commensal organisms in the gut or mouth that may shape immune responses in RA. Using the same discovery-based proteomic and translational research approach developed for studies of Lyme arthritis, we recently identified T cell epitopes of a gut commensal microbe, Prevotella copri and two novel autoantigens, N-acetylglucosamine-6-sulfatase (GNS) and filamin A (FLNA) in patients with RA. Moreover, we then showed that approximately half of RA patients had T and/or B cell reactivity with these microbial and self-proteins.

Hypotheses Connecting Mucosal Immunity and Autoimmunity in Joints in RA
Two possible mechanisms were identified that may explain a gut-joint connection. First, we demonstrated cross-reactive T cell epitopes of Prevotella sp. and GNS and FLNA epitopes, suggesting that molecular mimicry may be a potential link between gut microbial immunity and autoimmunity in joints. Second, in patients with IgG P. copri antibody responses, we identified HLA-DR-presented P. copri peptides in their PBMC and Prevotella DNA in their synovial fluid, suggesting that the organism may spread systemically, presumably within cells, sometimes reaching joints. In collaboration with Dr. Noah Palm at Yale University, we plan to identify other immunorelevant pathobionts in patients’ stool samples using flow cytometry and 16S sequencing.

In addition to the gut-joint connection, periodontitis and immune responses to several periodontal pathogens, such as Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans (Aa), have been associated with RA. The latter organism has a leukotoxin with lyses PMNs and causes them to release an enzyme which citrullinates host proteins. This may lead to the development of anti-citrullinated protein antibodies (ACPA), which are a central feature of immune responses in RA patients. Together with investigators at the Harvard-affiliated Forsyth Institute, we are studying organisms in gingival crevicular fluid, immune responses to implicated periodontal pathogens and inflammatory responses in the mouth and joints, as with gut associated immune responses.

Diagnostic Tests and Treatment Studies

These human studies have important implications for the diagnosis and treatment of these diseases. Previously, this laboratory developed a serologic approach of ELISA and Western blot for the diagnosis of Lyme disease patients. We are continuing to improve tests for this infection and for the identification of patients at greater risk for adverse outcomes. This information will likely impact treatment and has already contributed to a treatment algorithm for antibiotic-refractory LA.

Our RA studies are now unfolding with a similar trajectory that includes the development of serologic and direct detection assays. Moreover, the most important long-term implication of this work is that it may lead to a new RA treatment paradigm. Targeting a specific community of microbes at the primary site of infection-induced autoimmune development would be associated with few, if any, side effects, and identification of pathogenic T cell epitopes may eventually make it possible to engineer blocking peptides to limit disadvantageous autoimmune responses. We are working on this provocative, but potentially transformative research, with the larger group of investigators in this field and training young scientists who will carry such studies into the next generation.

Research Projects

The projects listed below are often done in parallel in patients with Lyme arthritis or rheumatoid arthritis.

  • Identification of HLA-DR-peptide epitopes from microbial or self-proteins in synovial tissue or PBMC and testing of implicated peptides for T and B cell responses
  • Evaluation of implicated T cells for molecular mimicry between homologous microbial antigens and self-proteins by cloning and stimulating cells
  • Assessment of antibody cross-reactivity using recombinant microbial and host proteins and affinity-purified antibodies and determination of antibody phenotypes and function based on Fc receptor binding, IgG subclass, binding affinity and glycan profile
  • Characterization of immune cell phenotypes by flow cytometry and single cell RNA-seq.
  • Evaluation of host and microbial genetic factors that contribute to dysregulated immunity and adverse patient outcomes
  • Determination of biomarkers, serologic tests and direct detection assays for improvement of patient diagnosis and treatment

Many of these projects are done in collaboration with investigators in our Center for Immunology and Inflammatory Diseases, and with those in surrounding institutions, including the Broad Institute, the Ragon Institute, the Forsythe Institute, and the Boston University Biomedical Mass Spectrometry Center.

Research Positions

Support for these studies and future plans

The laboratory is supported by two NIH R01 grants, which were recently funded for five years, an R21 grant, as well as grants from three private foundations. Several post-doctoral fellows have recently graduated from our program, and we are currently recruiting to replace them. Thus, our plan is to double the number of post-doctoral fellows and technicians during the coming year.


Lab Members

Principal Investigator

Allan Steere, MD

Allen Steere, MD
Physician, Department of Medicine, Massachusetts General Hospital
Director of Translational Research, Rheumatology Unit, Massachusetts General Hospital
Professor of Medicine, Harvard Medical School


Co-Investigator

Klemen Strle, PhD
Co-Investigator, Center for Immunology and Inflammatory Diseases, Massachusetts General Hospital
Assistant Professor of Medicine, Harvard Medical School

Research Team

Sheila Arvikar, MD, Instructor
David Ordonez Del Valle, PhD, Postdoctoral Fellow
Geena Chiumenta, Research Technician

Publications

Selected Publications

Discovery and elucidation of Lyme disease

Steere AC, Malawista SE, Snydman DR, Shope RE, Andiman WA, Ross MR, Steele FM. Lyme arthritis: an epidemic of oligoarticular arthritis in children and adults in three Connecticut communities. Arthritis Rheum 1977; 20:7-17.

Steere AC, Malawista SE, Hardin JA, Ruddy S, Askenase PW, Andiman WA. Erythema chronicum migrans and Lyme arthritis: the enlarging clinical spectrum. Ann Intern Med 1977; 86:685-98.

Steere AC, Malawista SE. Cases of Lyme disease in the United States: locations correlated with distribution of Ixodes dammini. Ann Intern Med 1979;91:730-3.

Steere AC, Grodzicki RL, Kornblatt AN, Craft JE, Barbour AG, Burgdorfer W, Schmid GP, Johnson E, Malawista SE. The spirochetal etiology of Lyme disease. N Engl J Med 1983; 308:733-40.

Development of diagnostic tests and appropriate antibiotic treatment regimens for Lyme disease

Dressler F, Whalen JA, Reinhardt BN, Steere AC. Western blotting in the serodiagnosis of Lyme disease. J Infect Dis 1993; 167:392-400.

Nocton JJ, Dressler F, Rutledge BJ, Rys PN, Persing DH, Steere AC. Detection of Borrelia burgdorferi DNA by polymerase chain reaction in synovial fluid in Lyme arthritis. N Engl J Med 1994; 330:229-34.

Steere AC, Green J, Schoen RT, Taylor E, Hutchinson GJ, Rahn DW, Malawista SE. Successful parenteral penicillin therapy of established Lyme arthritis. N Engl J Med 1985; 312:869-74.

Steere AC, Levin RE, Molloy PJ, Kalish RA, Abraham JH III, Liu NY, Schmid CH. Treatment of Lyme arthritis. Arthritis Rheum 1994; 37:878-88.

Development and testing of a Lyme disease vaccine

Steere AC, Sikand VK, Meurice F, Parenti DL, Fikrig E, Schoen RT, Nowakowski J, Schmid CH, Laukamp S, Buscarino C, Krause DS, and the Lyme Disease Vaccine Study Group. Vaccination against Lyme disease with recombinant Borrelia burgdorferi outer-surface lipoprotein A with adjuvant. N Engl J Med 1998; 339:209-15.

Elucidation of pathogenetic factors and treatment for post-infectious, antibiotic-refractory Lyme arthritis.

Steere AC, Klitz W, Drouin EE, Falk BA, Kwok WW, Nepom GT, Baxter-Lowe LA. Antibiotic-refractory Lyme arthritis is associated with HLA-DR molecules that bind a Borrelia burgdorferi peptide. J Exp Med 2006;203:961-71.

Strle K, Shin JJ, Glickstein LJ, Steere AC. A toll-like receptor 1 polymorphism is associated with heightened T-helper 1 responses and antibiotic-refractory Lyme arthritis. Arthritis Rheum 2012;64:1497-1507. PMCID:PMC 3338893

Drouin EE, Seward RJ, Strle K, McHugh G, Katchar K, Londono D, Yao C, Costello CE, Steere AC. A novel human autoantigen, endothelial cell growth factor, is a target of T and B cells responses in patients with Lyme disease. Arthritis Rheum 2013;65:186-196. PMCID:PMC 3535550

Crowley JT, Strle K, Drouin EE, Pianta A, Arvikar SL, Wang Q, Costello CE, Steere AC. Matrix metalloproteinase-10 is a target of T and B cell responses that correlate with synovial pathology in patients with antibiotic-refractory Lyme arthritis. J Autoimmun 2016;5,69:24-37. PMCID:PMC 4826816

Identification of Prevotella copri and related autoantigens as having immune relevance in patients with rheumatoid arthritis.

Seward R, Drouin EE, Steere AC, Costello C. Peptides presented by HLA-DR molecules in synovia of patients with rheumatoid arthritis or antibiotic-refractory Lyme arthritis. Mol Cell Proteomics 2011;10:M110002477. PMCID:PMC 3047150

Wang Q, Drouin EE, Yao C, Zhang J, Huang Y, Leon DR, Steere AC, Costello CE. Immunogenic HLA-DR-presented self-peptides identified directly from clinical samples of synovial tissue, synovial fluid, or peripheral blood in patients with rheumatoid arthritis or Lyme arthritis. J Proteome Res 2017;16:122-136. PMID:PMC 5766322

Pianta A, Arvikar SL, Strle K, Drouin EE, Wang Q, Costello CE, Steere AC. Evidence for immune relevance of Prevotella copri, a gut microbe, in patients with rheumatoid arthritis. Arthritis Rheumatol 2017;69:964-75. PMCID:PMC 5406252

Pianta A, Arvikar SL, Strle K, Drouin EE, Wang Q, Costello CE, Steere AC. Two rheumatoid arthritis-specific autoantigens correlate microbial immunity with autoimmune responses in joints. J Clin Invest 2017;127:2946-56. PMCID:PMC 5531397

Review articles

Steere AC, Strle F, Wormser GP, Hu LT, Branda JA, Hovius JWR, Li X, Mead PS. Lyme borreliosis. Nature Rev Dis Primer 2016 Dec 15;2:16090. PMCID:PMC 5539539.

Steere AC, Glickstein L. Elucidation of Lyme arthritis. Nature Rev Immunol 2004;4:143-152

Steere AC, Coburn J, Glickstein L. The emergence of Lyme disease. J Clin Invest 2004:113:1093-1101.

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