Faculty Bibliography

Background/Purpose: A transmissible agent has long been suspected in SLE. In a discovery cohort we found that,compared with healthy subjects, Lupus patients had a five-fold overall mean greater representation of the Ruminococcus gnavus species of the Lachnospiraceae family of anaerobic gram-positive cocci. Many SLE patients also displayed biomarkers of increased gut permeability that has been associated with bacterial translocation. In a patient, the relative fecal R. gnavus abundance directly correlated with serum levels of IgG anti-R. gnavus antibodies. This antibacterial immune response had features indicative of molecular mimicry with anti-native DNA antibodies, which are direct contributors to the pathogenesis of Lupus nephritis (LN). We therefore sought to isolate and characterize the responsible strain-associated antigen in the R. gnavus candidate pathobiont.
Method(s): A panel of strains of the Lachnospiraceae family, and other anaerobic commensals, were evaluated by immunoblot, bead-based Luminex assay, as well as direct and competition ELISA. To isolate lipoconjugates from the candidate Gram-positive bacterial commensal strain, we applied a validated extraction protocol using a butanol separation with fractionation after passage over Hydrophobic Interaction Chromatography (HIC). Fractions were then evaluated for immunoreactivity and capacity to stimulate a human TLR2- transfected HEK reporter gene system.
Result(s): In an assay with a cutoff based on the mean+2SD of 55 healthy controls, we found that one of 8 independent R. gnavus isolates, which we termed RG2, displayed high level serum IgG reactivity with 30 of 50 active LN patients and only 4 of 36 patients without active renal disease (P<0.001 by t test). Fractionation of the RG2 extract by chromatography enabled the isolation of a lipoglycan, which by high-resolution NMR spectroscopy showed the presence of repetitive oligosaccharide building blocks. Sera from LN patients also displayed strong IgG reactivity with the lipoglycan, which was inhibitable by the nuclease-treated RG2 extract. In side-by-side analyses the lipoglycan had the same oligomeric immunoblot banding pattern of the parental bacterial extract, and in a HEK transfected reporter cell system displayed strong dose-dependent (range 2 to 2000 ng/ml) NFkB activation, compared to nonstimulated conditions. This activity was significantly inhibited by an anti-huTLR2 mAb at 10 ug/ml (P=0.006) but not isotype control. Further analyses of the exact carbohydrate content, as well as the overall size and detailed structure of the isolated lipoglycan, are currently under study by NMR, MS, and GC/MS.
Conclusion(s): We have identified a gut commensal strain-associated cell wall lipoglycan, which appears to represent an immunodominant antigen in a candidate pathobiont implicated in the pathogenesis of LN. Moreover, the TLR2-activation potential of the lipoglycan pool may be due to the presence of lipopeptides, which will require further investigation. Our findings suggest a pathway by which continuous translocation of a bacterial mimic of DNA with innate immunostimulatory properties may contribute to the pathogenesis of LN

Background A transmissible agent has long been suspected inthe pathogenesis of SLE, yet the potential contribution of thehuman intestinal microbiome has been little examined. Wetherefore characterized the gut microbiota of patients withSLE, with special interest in those with lupus nephritis (LN).Methods Blood and fecal samples from SLE patients wereobtained, with strict inclusion/exclusion of criteria. Fecal 16SrDNA sequencing, as well as cytokine and autoantibody assayswere performed. In addition, sera from two independent lupuscohorts were studied for validation. Biomarkers of gut leakiness were assessed.Results Compared to controls, the intestinal microbiome fromSLE patients (n=61) showed decreased species richness diversity with reductions in taxonomic complexity mostpronounced in those with high disease activity. Notably, SLEpatients had an overall 5-fold greater representation of a species in the Lachnospiracaea family of obligate anaerobic Grampositive cocci, with reciprocal contractions of two other commensal species with putative protective properties. Abundanceof the Lachnospiracaea species correlated with serum IgG to acell wall component, postulated to represent a lipoglycan,from a strain of this same species (p=0.002, n=61, Spearman)but not with 7 other strains. There was also a significantdirect correlation between SLEDAI scores and levels of thesecirculating anti-strain IgG antibodies (p=0.02, n=48). Levelsof antibodies to strain-specific bacterial antigen, treated withRNAse/DNAse/proteinase K, were significantly higher in thosewith active nephritis at time of sampling compared to SLEwithout renal activity (Cohort 1 p=0.01 n=48; Cohort 2p=0.006, n=28, Mann-Whitney). Levels of serum IgG antistrain antibodies also significantly correlated with high-titerserum IgG to native DNA (p<0.0001, n=27), and inverselycorrelated with C3 and C4 levels. High titers of these antibacterial antibodies were associated with active Class III, IVand V (overlap) LN (Cohort 3).Conclusions These findings suggest a novel paradigm for thepathogenesis of LN in which a common intestinal commensalbacteria may contribute to the immune-complex mediated disease process, with features akin to poststreptococcal GN butwithout outward signs and symptoms of clinical infection

BACKGROUND:Immunoglobulin M (IgM) autoreactivity to malondialdehyde (MDA) protein modifications is part of the natural antibody repertoire in health and may have beneficial functions. In contrast, IgG anti-MDA are increased in chronic inflammation and autoimmunity and may instead have pathogenic properties. METHODS:Herein, we investigated serum IgG anti-MDA levels by enzyme-linked immunosorbent assay (ELISA) in 398 systemic lupus erythematosus (SLE) patients in the Swedish Karolinska SLE cohort and compared these to findings in 225 US SLE patients from New York University and Johns Hopkins University. RESULTS:In two independent cohorts, IgG anti-MDA levels correlated positively with disease activity by the Systemic Lupus Erythematosus Disease Activity Index (SLEDAI; p < 0.0001, Spearman R = 0.3). Meta-analysis found an odds ratio of 2.7 (confidence interval (CI) 1.9-3.9; p < 0.0001) for high anti-MDA IgG levels with active disease (SLEDAI ≥ 6). Furthermore, IgG anti-MDA correlated directly with erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), soluble tumor necrosis factor receptors (sTNFR-1, sTNFR-2), and vascular cell adhesion molecule 1 (VCAM-1) measurements, and inversely with complement factors (C1q, C2, C3, C4). Importantly, IgG anti-MDA levels were significantly elevated in SLE patients with active nephritis (p = 0.0005) and correlated with cystatin C estimated glomerular filtration rate and albuminuria. CONCLUSIONS:Elevated IgG anti-MDA in SLE patients was associated with high disease activity, with active lupus nephritis, and with biomarkers of systemic inflammation. This natural antibody reactivity may have potential prognostic utility, and may also actively contribute to pathogenesis.

Staphylococcus aureus is a common commensal and frequent opportunistic pathogen that causes invasive infections that often recur. Co-evolution with the host has led to the development of toxins that affect diverse immune cell types. Recent reports have highlighted the contributions of staphylococcal protein A (SpA). This small oligomeric secreted protein contains 4-5 homologous domains with two distinct immunoglobulin-binding sites; one for IgG Fc domains, while a separate site binds an evolutionarily conserved surface on Fab encoded by VHIII clan related genes. The Fab-binding site has been implicated in in vivo supraclonal VHIII-BCR targeted B-cell depletion by an activation induced death pathway. Yet the concept of a superantigen for B lymphocytes poses a seeming paradox. Unlike TCR that are expressed only in a membrane-associated form, BCR are expressed in both a membrane BCR form and in secreted Ig forms, which permeate virtually every part of the body at high levels. We therefore asked, why circulating immunoglobulin do not block the superantigen properties of SpA? Herein, we show that soluble IgG molecules are not in vivo inhibitors of these B-cell superantigen effects but are instead essential for potentiating these properties. We also show that the Fc subclass of circulating IgG is an indirect critical determinant of the B-cell superantigen effect. In contrast, host FcγR and complement are not required for SpA mediated in vivo B-cell depletion. Unexpectedly, after VHIII-IgG2a pretreatment SpA challenge resulted in fatal anaphylactic reactions, which we speculate may have involved FcγR interactions with mast cells and basophils. Cumulatively, our findings illuminate a cunning and potent molecular strategy by which a bacterial toxin effectively confounds the contributions of host B-lymphocytes to immune defenses.

Oxidation-associated malondialdehyde (MDA) modification of proteins can generate immunogenic neo-epitopes that are recognized by autoantibodies. In health, IgM antibodies to MDA-adducts are part of the natural antibody pool, while elevated levels of IgG anti-MDA antibodies are associated with inflammatory and autoimmune conditions. Yet, in human autoimmune disease IgG anti-MDA responses have not been well characterized and their potential contribution to disease pathogenesis is not known. Here, we investigate MDA-modifications and anti-MDA-modified protein autoreactivity in rheumatoid arthritis (RA). While RA is primarily associated with autoreactivity to citrullinated antigens, we also observed increases in serum IgG anti-MDA in RA patients compared to controls. IgG anti-MDA levels significantly correlated with disease activity by DAS28-ESR and serum TNF-alpha, IL-6, and CRP. Mass spectrometry analysis of RA synovial tissue identified MDA-modified proteins and revealed shared peptides between MDA-modified and citrullinated actin and vimentin. Furthermore, anti-MDA autoreactivity among synovial B cells was discovered when investigating recombinant monoclonal antibodies (mAbs) cloned from single B cells, and 3.5% of memory B cells and 2.3% of plasma cells were found to be anti-MDA positive. Several clones were highly specific for MDA-modification with no cross-reactivity to other antigen modifications such as citrullination, carbamylation or 4-HNE-carbonylation. The mAbs recognized MDA-adducts in a variety of proteins including albumin, histone 2B, fibrinogen and vimentin. Interestingly, the most reactive clone, originated from an IgG1-bearing memory B cell, was encoded by near germline variable genes, and showed similarity to previously reported natural IgM. Other anti-MDA clones display somatic hypermutations and lower reactivity. Importantly, these anti-MDA antibodies had significant in vitro functional properties and induced enhanced osteoclastogenesis, while the natural antibody related high-reactivity clone did not. We postulate that these may represent distinctly different facets of anti-MDA autoreactive responses.