Allen C. Steere

Objective: The “mucosal origins hypothesis” posits that mucosal sites, such as the gut, are initial sites where immune responses first develop that may, months to years later, result in clinical rheumatoid arthritis (RA). Our goal here was to conduct a discovery-based, hypothesis-generating study using samples from a relatively small number of patients to implicate previously unrecognized highly IgA-coated commensal flora that may have a role in RA pathogenesis (primary outcome). The secondary supporting outcomes were to determine whether HLA-DR-presented microbial peptides from such flora could be identified in synovial tissue and to determine T and B cell responses to these peptides and citrullinated proteins. Methods: Highly IgA-coated bacteria taxa were identified in RA patients’ stool samples by 16s rRNA sequencing; HLA-DR-presented peptides derived from the most common, highly IgA-coated microbes were identified from synovial tissue by liquid chromatography–tandem mass spectrometry; T cell responses to the identified microbial and human mimic epitopes were determined by IFNγ ELISpot assays, and citrullinated IgG and IgA antibodies to implicated microbial and human proteins were measured by ELISA. Results: Of 32 RA patients, 28 (88%) had highly IgA-coated Akkermansia muciniphila ( Am ) in stool samples. HLA-DR presentation of peptides derived from two Am proteins, including the molecular chaperone DnaK protein ( Am -cDnaK), was identified from RA synovial tissue, suggesting that the microbe or its components may reach the joints. Eight of 21 patients (38%) had T cell responses to the Am -cDnaK peptide and a human heat shock protein-mimic peptide (Hu-HSPA5) and/or IgG antibodies to citrullinated forms of these proteins. Reactivity was found primarily in pre-RA and early RA soon after transition to clinical RA and less commonly during later flares of arthritis. Conclusions: These data implicate Am as a candidate gut commensal involved in RA pathogenesis and may serve as a compass for planning larger studies to test the hypothesis that Am , as well as certain other highly IgA-coated commensal flora or their components, may sometimes reach joints where they initiate or enhance autoimmune T cell responses via epitope mimicry and epitope spreading, leading to antibody responses to citrullinated host proteins in joints.

Persistent symptoms after an acute infection is an emerging public health concern, but the pathobiology of such conditions is not well understood. One possible scenario involves the persistence of lingering antigen. We have previously reported that patients with postinfectious Lyme arthritis often harbor the peptidoglycan (PG) cell wall of Borrelia burgdorferi , the Lyme disease agent, in the synovial fluid of their inflamed joints after treatment. However, it is not yet known how B. burgdorferi PG persists, in what form, or if it may play a role in other postinfectious complications after Lyme disease. Using a murine model, we developed a real-time in vivo system to track B. burgdorferi PG as a function of cell wall chemistry and validated our findings using both molecular and cellular approaches. Unlike typical bacterial PG, the unique chemical properties of polymeric B. burgdorferi PG drive murine liver accumulation, where the cell wall material persists for weeks. Kupffer cells and hepatocytes phagocytose and retain B. burgdorferi PG and, although liver occupancy coincides with minimal pathology, both organ-specific and secreted protein profiles produced under these conditions bear some similarities to reported proteins enriched in patients with chronic illness after acute infection. Moreover, transcriptomic profiling indicated that B. burgdorferi PG affects energy metabolism in peripheral blood mononuclear cells. Our findings provide mechanistic insights into how a pathogenic molecule can persist after agent clearance, potentially contributing to illness after infection.

The spirochete Borrelia burgdorferi causes Lyme disease. In some patients, an excessive, dysregulated proinflammatory immune response can develop in joints leading to persistent arthritis even after antibiotic therapy. In such patients, persistence of antigenic B. burgdorferi peptidoglycan (PG ^Bb ) fragments within joint tissues may contribute to immunopathogenesis pre- and post-antibiotic treatment. In live B. burgdorferi cells, the outer membrane shields the polymeric PG ^Bb sacculus from exposure to the immune system. However, unlike most diderm bacteria, B. burgdorferi releases PG ^Bb turnover products into its environment due to the absence of recycling activity. In this study, we identified the released PG ^Bb fragments using a mass spectrometry-based approach. By characterizing the L,D-carboxypeptidase activity of B. burgdorferi protein BB0605 (renamed DacA), we found that PG ^Bb turnover largely occurs at sites of PG ^Bb synthesis. In parallel, we demonstrated that the lytic transglycosylase activity associated with BB0259 (renamed MltS) releases PG ^Bb fragments with 1,6-anhydro bond on their N -acetylmuramyl residues. Stimulation of human cell lines with various synthetic PG ^Bb fragments revealed that 1,6-anhydromuramyl-containing PG ^Bb fragments are poor inducers of a NOD2-dependent immune response relative to their hydrated counterparts found in the polymeric PG ^Bb isolated from dead bacteria. We also showed that the activity of the human N -acetylmuramyl-L-alanine amidase PGLYRP2, which reduces the immunogenicity of PG ^Bb material, is low in joint (synovial) fluids relative to serum. Altogether, our findings suggest that MltS activity helps B. burgdorferi evade PG-based immune detection by NOD2 during growth despite shedding PG ^Bb fragments and that PG ^Bb -induced immunopathology likely results from host sensing of PG ^Bb material from dead (lysed) spirochetes. Additionally, our results suggest the possibility that natural variation in PGLYRP2 activity may contribute to differences in susceptibility to PG-induced inflammation across tissues and individuals. Author summary: During bacterial infection, the presence of peptidoglycan– a polymeric element of bacterial cell walls–triggers a host inflammatory response. Although generally protective during acute phases, inflammation, when chronic, can contribute to disease development. Recent work has suggested that the persistence of pro-inflammatory peptidoglycan derived from the Lyme disease spirochete Borrelia burgdorferi in joints may contribute to persistent arthritis in some patients despite appropriate antibiotic therapy. Interestingly, B. burgdorferi sheds peptidoglycan turnover products into the environment during growth. Here, we show that these shed products from live spirochetes are poor effectors of an immune response by the human NOD2 immune receptor due to the formation of an anhydro bond on the N -acetyl-muramic residue during peptidoglycan hydrolysis by a B. burgdorferi lytic transglycosylase. We also show that human N -acetylmuramyl-L-alanine amidase activity, which abrogates the NOD2-dependent response to the immunogenic peptidoglycan isolated from lysed B. burgdorferi cells, is low in human joint fluids relative to serum. Based on our findings, we propose that immunopathogenesis by peptidoglycan material more likely derives from lysed spirochetes (killed by an immune attack or antibiotics) than live ones and that the level of human peptidoglycan hydrolytic enzymes across tissues and individuals influences susceptibility to chronic inflammation.

Rheumatoid arthritis (RA) is a potentially devastating autoimmune disease. The great majority of patients with RA are seropositive for anti-citrullinated protein antibodies (ACPAs), rheumatoid factors, or other autoantibodies. The onset of clinically apparent inflammatory arthritis meeting classification criteria (clinical RA) is preceded by ACPA seropositivity for an average of 3–5 years, a period that is designated as ‘at-risk’ of RA for ACPA-positive individuals who do not display signs of arthritis, or ‘pre-RA’ for individuals who are known to have progressed to developing clinical RA. Prior studies of individuals at-risk of RA have associated pulmonary mucosal inflammation with local production of ACPAs and rheumatoid factors, leading to development of the ‘mucosal origins hypothesis’. Recent work now suggests the presence of multiple distinct mucosal site-specific mechanisms that drive RA evolution. Indicatively, subsets of individuals at-risk of RA and patients with RA harbour a faecal bacterial strain that has exhibited arthritogenic activity in animal models and that favours T helper 17 (T_H17) cell responses in patients. Periodontal inflammation and oral microbiota have also been suggested to promote the development of arthritis through breaches in the mucosal barrier. Herein, we argue that mucosal sites and their associated microbial strains can contribute to RA evolution via distinct pathogenic mechanisms, which can be considered causal mucosal endotypes. Future therapies instituted for prevention in the at-risk period, or, perhaps, during clinical RA as therapeutics for active arthritis, will possibly have to address these individual mechanisms as part of precision medicine approaches.

Lyme disease, caused by Borrelia burgdorferi ( Bb ), can progress to Lyme arthritis (LA). While most patients with LA respond successfully to antibiotic therapy, a small percentage fail to improve, a condition known as antibiotic-refractory Lyme arthritis (ARLA). While T cell responses are known to drive ARLA, molecular mechanisms for ARLA remain unknown. In this issue of the JCI , Dirks et al. isolated disease-specific Th cells from patients with ARLA residing in Germany. A distinct TCR-β motif distinguished ARLA from other rheumatic diseases. Notably, the TCR-β motif was linked predominantly to HLA-DRB1*11 or 13 alleles, which differed from alleles in patients from North America. It also mapped primarily to T peripheral helper (Tph) cells, as opposed to classical Th1 cells. These findings provide a roadmap explaining how T cell responses necessary for control of an infection can, despite antibiotic therapy, drive a disadvantageous T cell response, resulting in a postinfectious, inflammatory arthritis.

Lyme arthritis (LA) was recognized as a separate entity in 1975 because of geographic clustering of children often diagnosed with juvenile rheumatoid arthritis in Lyme, Connecticut. After identification of erythema migrans as a common early feature of the illness, a prospective study of such patients implicated Ixodes scapularis ticks in disease transmission. In 1982, the causative agent, now called Borrelia burgdorferi, was cultured from these ticks and from Lyme disease patients. Subsequently, it was shown that LA could usually be treated successfully with oral antibiotics but sometimes required intravenous antibiotics. Yet, a small percentage of patients developed a dysregulated, proinflammatory immune response leading to persistent postinfectious synovitis with vascular damage, cytotoxic and autoimmune responses, and fibroblast proliferation, a lesion similar to that of rheumatoid arthritis. The message from postinfectious LA for other autoimmune arthritides is that a complex immune response with autoimmune features can begin with a microbial infection.

Objective: HLA‐DR–expressing fibroblast‐like synoviocytes (FLS) are a prominent cell type in synovial tissue in chronic inflammatory forms of arthritis. FLS‐derived extracellular matrix (ECM) proteins, including fibronectin‐1 (FN1), contain immunogenic CD4+ T cell epitopes in patients with postinfectious Lyme arthritis (LA). However, the role of FLS in presentation of these T cell epitopes remains uncertain.Methods: Primary LA FLS and primary murine FLS stimulated with interferon gamma (IFNγ), Borrelia burgdorferi, and/or B burgdorferi peptidoglycan (PG) were assessed for properties associated with antigen presentation. HLA‐DR–presented peptides from stimulated LA FLS were identified by immunopeptidomics analysis. OT‐II T cells were co‐cultured with stimulated murine FLS in the presence of cognate ovalbumin antigen to determine the potential of FLS to act as inducible antigen presenting cells (APCs).Results: FLS expressed HLA‐DR molecules within inflamed synovial tissue and tendons from patients with postinfectious LA in situ. Major histocompatibility complex (MHC) class II and co‐stimulatory molecules were expressed by FLS following in vitro stimulation with IFNγ and B burgdorferi and presented both foreign and self‐MHC‐II peptides, including an immunogenic T cell epitope derived from Lyme autoantigen FN1. Stimulated FLS induced proliferation of naive OT‐II CD4+ T cells that were dependent on OT‐II antigen and CD40. Stimulation with B burgdorferi PG enhanced FLS‐mediated T cell activation.Conclusion: MHC‐II+ FLS are inducible APCs that can induce CD4+ T cell activation in an antigen‐ and CD40‐dependent manner. Activated FLS can also present ECM‐derived Lyme autoantigens, implicating FLS in amplifying tissue‐localized autoimmunity in LA.

Background: HLA-DR-expressing fibroblast-like synoviocytes (FLS) are a prominent cell type in synovial tissue in chronic inflammatory forms of arthritis. We recently showed that peptides from several extracellular matrix (ECM) proteins, including fibronectin-1 (FN1), contained immunogenic CD4+ T cell epitopes in patients with postinfectious Lyme arthritis (LA). However, the role of FLS in presentation of these T cell epitopes remains uncertain. Methods: Primary LA FLS and primary murine FLS stimulated with interferon gamma (IFNγ), Borrelia burgdorferi , and/or B. burgdorferi peptidoglycan (PG) were assessed for properties associated with antigen presentation. HLA-DR-presented peptides from stimulated LA FLS were identified by immunopeptidomics analysis. OT-II T cells were cocultured with stimulated murine FLS in the presence of cognate ovalbumin antigen to determine the potential of FLS to act as inducible antigen presenting cells (APC). Results: FLS expressed HLA-DR molecules within inflamed synovial tissue and tendons from patients with post-infectious LA patients in situ. MHC class II and costimulatory molecules were expressed by FLS following in vitro stimulation with IFNγ and B. burgdorferi and presented both foreign and self MHC-II peptides, including T cell epitopes derived from two Lyme autoantigens fibronectin-1 (FN1) and endothelial cell growth factor (ECGF). Stimulated murine FLS induced proliferation of naïve OT-II CD4+ T cells, particularly when FLS were stimulated with both IFNγ and PG. Conclusions: MHC-II+ FLS are inducible APCs that can induce CD4+ T cell activation and can present Lyme autoantigens derived from ECM proteins, thereby amplifying tissue-localized autoimmune CD4+ T cell responses in LA. AUTHORS’ SUMMARY: This study demonstrates that IFNγ-activated MHC-II+ fibroblast-like synoviocytes (FLS) stimulated with Borrelia burgdorferi present foreign and self MHC-II antigens, including Lyme autoantigens. Furthermore, IFNγ-activated MHC-II+ FLS stimulated with B. burgdorferi peptidoglycan can induce activation and proliferation of naïve CD4+ T cells in an MHC-II antigen-dependent manner, demonstrating that activated MHC-II+ FLS are inducible antigen presenting cells.