Faculty Bibliography

Background: A transmissible agent has long been suspected in the pathogenesis of SLE. We therefore investigated the potential contribution of the intestinal microbiome to LN.
Method(s): Blood and fecal samples from SLE patients were obtained, unless a patient had selective IgA deficiency, prior cytotoxic drugs, or antibiotics within four months. Fecal 16S rRNA NGS was performed. Sera samples were profiled for autoantibodies. Sera from two independent lupus cohorts were studied for validation.
Result(s): Compared to controls, the intestinal microbiome from SLE patients (N=61) showed decreased species richness diversity. The microbiomes of patients in clinical remission (based on SLEDAI) were most similar to healthy controls, while reductions in taxonomic complexity were most pronounced in those with high disease activity. Notably, SLE patients had an overall 5-fold greater representation of a particular species in the Blautia genus of the Lachnospiracaea family of obligate anaerobic Gram-positive cocci. Abundance of this species significantly correlated with serum IgG to a cell wall moiety from a strain of this species (P=0.002, N=61, Spearman) but not with 7 other strains. There was also a significant correlation between the distribution of SLEDAI scores and levels of these circulating anti-strain IgG antibodies (P=0.02, N=48). Using antigen treated with DNAse/proteinase K, levels of IgG anti-strain antibodies were significantly higher in those with active nephritis at time of sampling compared to SLE without renal activity (Cohort 1 P=0.01 N=48; Cohort 2 P=0.001, N=53, Mann-Whitney). Levels of anti-strain antibodies also significantly correlated with high-titer serum IgG to native DNA (P<0.0001, N=27), and inversely correlated with C3 and C4 (each P<0.01, N=61). High titers of these anti-bacterial antibodies were found in active Class III, IV and V LN.
Conclusion(s): These findings suggest a novel paradigm for the pathogenesis of LN: Specific strains of common intestinal commensal bacteria affect IgG-autoantibody responses in patients with LN. This is reminiscent of post-streptococcal GN, although the postulated intestinal bacterial bloom occurs without clinical infection

Natural IgM autoantibodies have been proposed to convey protection from autoimmune pathogenesis. Herein, we investigated the IgM responses in 396 systemic lupus erythematosus (SLE) patients, divided into subgroups based on distinct autoantibody profiles. Depressed IgM levels were more common in SLE than in matched population controls. Strikingly, an autoreactivity profile defined by IgG anti-Ro/La was associated with reduced levels of specific natural IgM anti-phosphorylcholine (PC) antigens and anti-malondialdehyde (MDA) modified-protein, as well total IgM, while no differences were detected in SLE patients with an autoreactivity profile defined by anti-cardiolipin/beta2glycoprotein-I. We also observed an association of reduced IgM levels with the HLA-DRB1*03 allelic variant among SLE patients and controls. Associations of low IgM anti-PC with cardiovascular disease were primarily found in patients without antiphospholipid antibodies. These studies further highlight the clinical relevance of depressed IgM, and suggest that low IgM levels in a SLE patient may reflect underlying immunological differences.

Since the introduction of TNF-alpha inhibitors and other biologic agents, the clinical outcome for many treated rheumatoid arthritis patients has significantly improved. However, there are still a substantial proportion of patients that are intolerant, or have inadequate responses, with current agents that have become the standards of care. While the majority of these agents are designed to affect the inflammatory features of the disease, there are also agents in the clinic that instead target lymphocyte subsets (e.g., rituximab) or interfere with lymphocyte co-receptor signaling pathways (e.g., abatacept). Due in part to their ability to orchestrate downstream inflammatory responses that lead to joint damage and disease progression, pathogenic expansions of T and B lymphocytes are appreciated to play key roles in the pathogenesis of rheumatoid arthritis. New insights into immune regulation have suggested novel approaches for the pharmacotherapeutic targeting of lymphocytes. In this review, we discuss deepening insights into human genetics and our understanding of the interface with rheumatoid arthritis pathogenesis providing a strong rationale for exploiting the co-inhibitory receptor programmed cell death-1 signaling pathway as a better approach for the treatment of this chronic, often progressive destructive joint disease.

OBJECTIVE: In RA, autoreactive B cells are pathogenic drivers and sources of anti-citrullinated protein antibodies (ACPA) that serve as a diagnostic biomarker and predictor of worse long-term prognosis. Yet the immunobiologic significance of persistent ACPA production at a cellular level is poorly understood. METHODS: In a cross-sectional study of RA patients, we investigated for the presence of continued defects in immune homeostasis as a function of disease activity. Using an ELISA and a sensitive multiplex bead-based immunoassay, we characterized fine-binding antibody-specificities in sera, synovial fluid (SF) and B-cell culture supernatants. In this manner, we determined the frequency and epitope reactivity patterns of ACPA produced by SF B cells and switched-memory blood B cells, and compared the latter to serum ACPA levels and disease activity scores. RESULTS: Cultured B cells from SF were shown to spontaneously secrete ACPA, while constitutive IgG-autoantibody production by PBMC was substantially less frequent. After in vitro stimulation, PBMC secreted IgG ACPA that was overwhelmingly from switched-memory B-cells, across all patient groups treated with MTX and/or a TNF-inhibitor. Intriguingly, frequencies of ACPA-producing switched-memory B cells significantly correlated with serum IgG anti-CCP3 (r=0.57, p=0.003). Moreover, treatment-induced clinical remission had little or no effect on the circulating burden of switched-memory ACPA-producing B cells, in part explaining the continued dysregulation of humoral immunity. CONCLUSIONS: Our findings rationalize why therapeutic cessation most often results in disease reactivation and clinical flare. Hence, a clinical disease activity score is not a reliable indicator of the resolution of pathologic recirculating B-cell autoimmunity

For investigations of human B-cell receptor (BCR) repertoires, we have developed a protocol for large-scale surveys of human antibody heavy chain (VH) rearrangements. Here we study IgA repertoires, as more IgA antibodies are synthesized in the human body on a daily level than all other isotypes combined. In fact, IgA is secreted at all mucosal surfaces, and it is also secreted in the perspiration that coats our cutaneous surfaces. In these studies we can characterize the IgA clonal diversity of B-cell populations obtained from any donor. To recover representative repertoire libraries, we make our libraries from antibody gene transcript templates (i.e., cDNA), as these are closer reflections of the immune repertoire expressed at the antibody protein level. To avoid biases potentially introduced by upstream oligonucleotide primers that hybridize to variable region framework regions, our approach also uses rapid amplification of cDNA ends (RACE) of antibody transcripts. For exploration of human IgA responses, we have designed a duplexing antisense constant region primer that efficiently amplifies, side-by-side, heavy chain transcripts of both the IgA1 and IgA2 subclasses. By these methods we have begun to define the molecular differences in the IgA1 and IgA2 responses occurring simultaneously in different donors. These methods will be used to investigate the effects of microbial virulence factors on host defenses, during autoimmune responses, and in B-cell malignancies.

B cell development is a tightly regulated process dependent on sequential rearrangements of immunoglobulin loci that encode the antigen receptor. To elucidate the role of microRNAs (miRNAs) in the orchestration of B cell development, we ablated all miRNAs at the earliest stage of B cell development by conditionally targeting the enzymes critical for RNAi in early B cell precursors. Absence of any one of these enzymes led to a block at the pro- to pre-B cell transition due to increased apoptosis and a failure of pre-B cells to proliferate. Expression of a Bcl2 transgene allowed for partial rescue of B cell development, however, the majority of the rescued B cells had low surface immunoglobulin expression with evidence of ongoing light chain editing. Our analysis revealed that miRNAs are critical for the regulation of the PTEN-AKT-FOXO1 pathway that in turn controls Rag expression during B cell development.

Background/Purpose: SLE is a complex multifactorial systemic autoimmune disease, which has been attributed to poorly understood interactions between genetic and environmental factors. Recent reports have begun to elucidate how imbalances within intestinal communities of commensal bacteria may lead to triggering of inflammatory and autoimmune conditions. Our studies are designed to shed light on the interactions of the immune system and the gut microbiome that may contribute to lupus pathogenesis. Methods: We have assembled a cohort of 60 female SLE patients and matched 20 healthy controls, and biobanked blood and stool samples. DNA was then extracted from fecal bacterial samples, and from sorted endogenous IgA-coated and non-coated bacterial fractions. 16S bacterial rRNA gene sequencing was then performed by illumina NGS technology. Fecal and serum total Igs and autoantibodies were measured by ELISA and by multiplex-bead autoantigen assays. Results: Our analyses showed that SLE patients have significantly reduced diversity (i.e., number of different taxa) in their gut microbiomes compared to controls (p=0.038). This dysbiosis was treatment independent, with more marked contractions in patients with high disease activity, based on SLEDAI (p=0.002). The distribution of microbiome taxa was more heterogeneous among SLE patients than healthy individuals (p=0.002). Interestingly, patients with the most active disease commonly displayed expansions of genus and species with putative pathobiont properties, with reciprocal contractions of others associated with protective properties (e.g. R. gnavus (p= 0.001) vs. F. prausnitzii (p= 0.022) and B. uniformis (p=0.016). In immunologic surveys, we found that IgA (the most highly produced Ig isotype in the body), was significantly elevated in SLE patients compared to healthy subjects (p<0.002). Only a limited proportion of bacterial taxa are specifically coated by endogenous intestinal IgA. Yet, SLE patients with high disease activity displayed an increased abundance among IgA-coated taxa of Prevotella copri (p= 0.018), a species which has recently been linked to new-onset RA. In addition, intestinal IgA in SLE patients included high levels of antibodies to lupus autoantigens, with the same IgA autoantibody profiles in the matched sera of individual patients. Conclusion: Our studies document that SLE is associated with a dysbiosis in the gut microbiome with expansions of specific pathobiont bacteria and reciprocal contractions that may contribute to immune dysregulation. This imbalance was more significant in patients with high disease activity. Certain microbial taxa/species are preferentially recognized by the adaptive immune system of SLE patients and coated in vivo by intestinal IgA, and this correlated with elevated overall levels of fecal and serum IgA. Taken together, these data support the hypothesis that the pathogenesis of SLE may arise from imbalances in the gut microbiome and immune recognition of certain bacterial taxa