Faculty Bibliography

Background/Purpose : Systemic lupus erythematosus (SLE) is the archetypic systemic autoimmune disease, for which there is mounting evidence for roles for intestinal bacteria in the development of the systemic autoimmune responses, inflammation, and tissue injury. Whereas Lupus nephritis (LN) is amongst the most serious and potentially life-threatening complications of SLE, earlier diagnosis would speed clinical decision-making and initiation of definitive therapy. To characterize the interrelationships between Lupus host immunity and a candidate gut microbiome pathobiont, we have recently described an assay for detection of serum IgG responses to the commensal anaerobe, Ruminococcus gnavus ( RG ), for identification of individuals with active LN in three US adult Lupus cohort. In the current studies, we further validated this assay and investigated its potential utility in a Swedish cohort. Methods : Patients were recruited and characterized at the Karolinska Hospital, based on ACR criteria, with 38 LN confirmed by renal biopsy, 38 non-renal SLE patients, with 37 age-gender-ethnicity-matched unaffected population (Healthy) controls, 20 ANCA-associated vasculitis (AAV) and 14 IgA Nephropathy (IgAN) patients, based on renal biopsy and/or standard clinical criteria. At our NYU lab, serologic studies were performed with a previously described custom bead-based serologic assay with nuclease-treated RG2 extract and control analytes. Results : LN patients had significantly higher levels of IgG anti-RG2 than Healthy subjects (p< 0.0001,) other nonrenal Lupus patients (p=0.0002), or patients with IgAN (p< 0.0001) or AAV (p< 0.0001)(Wilcoxon rank-sum). There was a modest difference between non-renal Lupus patients, and Healthy subjects (p=0.0283), but no difference between Healthy with IgAN (p=0.273) or AAV (p=0.433). ROC analysis, when LN were compared to Healthy subjects gave an AUC of 0.863 and a Likelihood ratio (LR) of 5.6 at a cut-offof the mean+1 SD for control subjects (p< 0.0001); the LR was 5.8 for a cut-offof a mean+2SD (p< 0.0062), and LR of 7.8 for a cut-offof a mean+3SD (p=0.0284). For LN compared to the disease controls (AAV and IgAN) there was an AUC of 0.828 with a LR of 2.64 with a cut-offof a mean+1SD for disease controls (p=0.01), and the LR of 5.4 with a disease control mean+2SD (p=0.0071)(Figure 1A&B). Conclusion : These findings further validate that serum IgG anti-RG2 levels provide a robust biomarker for the identification of LN, unaffected by gender and age. Importantly, the assay displayed attractive performance characteristics Area under the curve estimates for comparisons of Lupus nephritis patients to A) age-and gender-matched population (Healthy) controls, or B) Disease controls with AAV or IgA nephropathy that represent other forms of glomerulopathy often complicated by pathologic levels of proteinuria. for discrimination of LN patients from population controls as well as those with pathologic proteinuria due to other forms of immune glomerulopathy (i.e., AAV and IgAN). Our findings also provide circumstantial evidence of a possible role for the R. gnavus pathobiont in patients in Sweden, suggesting this pathobiont may be involved in LN at sites geographically distant from the localities in which this association was first identified. (Figure Presented)

BACKGROUND/PURPOSE/OBJECTIVE:To search for a transmissible agent involved in lupus pathogenesis, we investigated the faecal microbiota of patients with systemic lupus erythematosus (SLE) for candidate pathobiont(s) and evaluated them for special relationships with host immunity. METHODS:In a cross-sectional discovery cohort, matched blood and faecal samples from 61 female patients with SLE were obtained. Faecal 16 S rRNA analyses were performed, and sera profiled for antibacterial and autoantibody responses, with findings validated in two independent lupus cohorts. RESULTS:strain-restricted antibodies were detected in those with active nephritis (including Class III and IV) in the discovery cohort, with findings validated in two independent cohorts. CONCLUSION/CONCLUSIONS:These findings suggest a novel paradigm in which specific strains of a gut commensal may contribute to the immune pathogenesis of lupus nephritis.

Background/UNASSIGNED:Patients with rheumatoid arthritis (RA) have an increased risk for cardiovascular disease. We examined the effect of gut microbiota in a mouse model of RA that develops atherosclerosis. Methods/UNASSIGNED:We created three groups of K/BxN female mice that were positive for the anti-glucose-6-phosphate isomerase (GPI) antibody: control diet (CD), high fat diet (HFD), and HFD with hydroxychloroquine (HFD + HCQ). Serological tests were used to detect the serum levels of total cholesterol (TCHO), low-density lipoprotein cholesterol (LDL-C), triglyceride (TG), high-density lipoprotein cholesterol (HDL-C), anti-GPI antibody titers, and serum cytokines. Atherosclerotic plaque was determined by histological analysis, and gut microbiota were determined by 16sV4 sequencing. Results/UNASSIGNED:cluster 1, and therefore may be responsible for the reduced RA-associated atherosclerosis and dyslipidemia. Conclusion/UNASSIGNED:Our mouse model of RA indicated that HFD increased ankle width and aggravated atherosclerosis and dyslipidemia, and that HCQ alleviated the dyslipidemia and atherosclerosis, but had no effect on ankle width.

T regulatory (Treg) cells maintain immunological tolerance and organ homeostasis. Activated Treg cells differentiate into effector Treg subsets that acquire tissue-specific functions. Ca²⁺ influx via Ca²⁺ release-activated Ca²⁺ (CRAC) channels formed by STIM and ORAI proteins is required for the thymic development of Treg cells, but its function in mature Treg cells remains unclear. Here we show that deletion of Stim1 and Stim2 genes in mature Treg cells abolishes Ca²⁺ signaling and prevents their differentiation into follicular Treg and tissue-resident Treg cells. Transcriptional profiling of STIM1/STIM2-deficient Treg cells reveals that Ca²⁺ signaling regulates transcription factors and signaling pathways that control the identity and effector differentiation of Treg cells. In the absence of STIM1/STIM2 in Treg cells, mice develop a broad spectrum of autoantibodies and fatal multiorgan inflammation. Our findings establish a critical role of CRAC channels in controlling lineage identity and effector functions of Treg cells.

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.

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