Faculty Bibliography

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/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

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.

Staphylococcus aureus is an opportunistic pathogen that causes a range of serious infections associated with significant morbidity, by strains increasingly resistant to antibiotics. However, to date all candidate vaccines have failed to induce protective immune responses in humans. We need a more comprehensive understanding of the antigenic targets important in the context of human infection. To investigate infection-associated immune responses, patients were sampled at initial presentation and during convalescence from three types of clinical infection; skin and soft tissue infection (SSTI), prosthetic joint infection (PJI) and pediatric hematogenous osteomyelitis (PHO). Reactivity of serum IgG was tested with an array of recombinant proteins, representing over 2,652 in-vitro-translated open reading frames (ORFs) from a community-acquired methicillin-resistant S. aureus USA300 strain. High-level reactivity was demonstrated for 104 proteins with serum IgG in all patient samples. Overall, high-level IgG-reactivity was most commonly directed against a subset of secreted proteins. Although based on limited surveys, we found subsets of S. aureus proteins with differential reactivity with serum samples from patients with different clinical syndromes. Together, our studies have revealed a hierarchy within the diverse proteins of the S. aureus "immunome", which will help to advance efforts to develop protective immunotherapeutic agents.