Faculty Bibliography

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: Malondialdehyde (MDA) is a naturally occurring reactive aldehyde that arises during apoptosis or as a consequence of elevated reactive oxygen species and lipid peroxidation. Free MDA can posttranslationally modify lysine residues in a protein carbonylation process that generates neo-epitopes that can be recognized by autoantibodies. Methods: Serum IgG anti-MDA levels were compared in 71 healthy controls, 30 OA, and 283 SLE and 162 RA patients, identified by ACR criteria. IgG anti-MDA was measured by sandwich ELISA using MDA-modified BSA. After flow cytometry sorting of RA synovial memory B cells, and single cell Ab-gene PCR, 114 mAbs were expressed and tested for MDA-reactivity. Analyses used the 2-sided Mann-Whitney test or Spearman correlation. Results: In sera, IgG anti-MDA was significantly increased in SLE (17+/-21 RU/ml, p<0.0001) and RA patients (13+/-11 RU/ml, p<0.0001), compared to controls (5+/-3 RU/ml). In SLE, IgG anti-MDA correlated with disease activity by SELENA-SLEDAI (p<0.0001, R=0.34, n=219). Levels were also significantly higher in SLE patients with active disease (SLEDAI>6, 18.9+/-17.3 RU/ml, p=0.001) than with low disease activity (SLEDAI<6, 11.5+/-16.6). In RA patients, IgG anti-MDA correlated with DAS28-ESR (p<0.0001, R=0.35, n=157). Compared to RA patients with low disease activity (DAS28<3.2, 6+/-3), levels were significantly increased in RA with moderate activity (DAS28 3.2-5.1, 12+/-11 RU/ml, p=0.005) and high activity (DAS28>5.1, 15+/-12 RU/ml, p=0.001). In DMARD naive RA (n=62), IgG anti-MDA also correlated with serum TNFalpha (p=0.002, R=0.39), IL-6 (p=0.03, R=0.27), and CRP levels (p=0.003, R=0.37). In RA synovial mAbs, we identified four clones (3.5%) that recognized MDA-modified epitopes. The most reactive clone, 1276:01F04, showed high specific binding to MDA but was non-reactive with carbamylated or 4-HNE-modifications. Specificity was confirmed in antigen-competition studies with MDA or MDA acetaldehyde (MAA) protein adducts. This mAb also bound MDA-modified human fibrinogen and albumin. No cross-reactivity with citrullinated epitopes was detected, and mAbs with ACPA or RF reactivity did not bind MDA. Notably, 1276:01F04 originated from an IgG1-bearing B cell that was encoded by near germline variable genes (VH4-39, 1 R-mutation in HCDR3; VL1-51, 2 S-mutations in FR1). Conclusion: IgM binding to MDA-adducts may be common in health from birth and are part of the natural antibody pool. Yet, IgG anti-MDA are associated with inflammatory conditions including increased disease activity in autoimmune patients. Importantly, MDA-autoreactive B cells could also be isolated from RA synovium, the site of disease. Further studies are merited to investigate the potential pathogenic properties of IgG anti-MDA B-cells/autoantibodies

Background/Purpose: Natural IgM autoantibodies have been proposed to have protective properties, and decreased levels of IgM to phosphorylcholine (PC) in SLE are associated with higher risk of atherosclerotic plaques and cardiovascular events. In the current study we investigated levels of total IgM and IgM anti-PC levels in different SLE subgroups. Methods: Serum IgM levels were compared in 322 population controls and 351 SLE patients meeting ACR criteria. SLE patients were subgrouped based on antibody profiles into antiphospholipid (APS)-like (n=54, with >2 positive tests among anti-CL/beta2GPI; LA), Sjogren's syndrome (SS)-like (n= 63, with >2 positive tests among anti-Ro52/Ro60/SSB), or SLE only with no "secondary syndrome" (n=234). A majority, but not all, of the APS-like and SS-like fulfilled clinical criteria for antiphospholipid syndrome or Sjogren's syndrome. Total IgM levels were determined in the clinical laboratory, and IgM anti-PC measured using PC-BSA by sandwich ELISA. Analyses used the 2-sided Mann-Whitney test or Spearman correlation. Results: IgM anti-PC levels were moderately but significantly decreased in the SLE patients (41.3+/-45.3 RU/ml) compared to matched controls (48.9+/-43.15 RU/ml) (n=316, p=0.002). Similarly, the total IgM levels were only slightly lower in SLE than controls (n=310, 1.22+/-1.1; 1.26+/-0.68 mg/ml, p=0.0002). There was a weak inverse correlation with age and disease duration for IgM anti-PC (p=0.02, R=-0.13; p=0.0003, R=-0.19). Importantly, APS-like patients did not have significantly altered total IgM (1.51+/-1.1 mg/ml) or IgM anti- PC levels (58.7+/-58.7 RU/ml) compared to controls (IgM 1.26+/-0.7 mg/ml; IgM anti-PC 48.7+/-43.2 RU/ml), while the SLE only group had moderately decreased IgM levels (IgM 1.21+/-1.3 mg/ml, p<0.0001; IgM anti-PC 39.5+/-43.0 RU/ml; p=0.002), and the SS-like patients had considerably impaired IgM (IgM 0.96+/-0.6 mg/ml, p<0.0001; IgM anti-PC 23.4+/-24.2 RU/ml, p<0.0001). There were no statistically significant differences based on age, disease duration, female sex, or anti-dsDNA positivity. In all SLE patients, total IgG levels were significantly increased compared to controls, and were highest in the SS-like group. Conclusion: Alterations in IgM levels between SLE subgroups further emphasize the immune heterogeneity of patients diagnosed with SLE. Patients have different clinical manifestations, risk of co-morbidities, and which also have distinct immunological features. Future studies are needed to investigate the clinical relevance of depressed IgM levels, and if the moderate IgM impairment in certain SLE patients reflects underlying immunological differences and/or is due to consumption of natural IgM or others causes resulting in skewing. of the B-cell repertoire

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

Background SLE is an archetypical systemic autoimmune disease,which has been attributed to interactions between genetic andenvironmental factors that are currently not well understood. Yetrecent reports have begun to elucidate how intestinal bacteriainfluence the development of physiologic B-cell/T-cell responses,and can affect the pathogenesis of inflammatory and autoimmuneconditions. Our studies are designed to shed light on the potential roles of the gut microbiome in SLE pathogenesis.Methods We have assembled and characterised a cohort of 60female SLE patients and 20 healthy controls. DNA from theunfractionated bacteria in faecal samples, and from the sortedendogenous IgA-coated and non-coated bacterial fractions, wasthen extracted. 16 S bacterial rRNA genes were then barcodedand amplified, and over 20,000 reads were determined per sample using illumina NGS technology. Faecal and serum total Ig andautoantibodies were measured by ELISA.Results Our analysis showed less microbiome diversity in SLEthan healthy controls (p = 0.002). This dysbiosis was treatmentindependent, with more severe intestinal dysbiosis and decreasedbacterial diversity in patients with high disease activity, based onSLEDAI. In addition, SLE patients had increased representationof certain bacterial families, genus's and species, based on 16 SrRNA assignments of operational taxonomic units (OTUs).Patients with active disease displayed contractions of bacterialtaxa with reported protective properties and reciprocal expansions of taxa with putative pathobiont properties. We alsoassessed IgA, which is the most prevalent antibody isotype madeby the human body, and found evidence of exuberant levels inboth intestinal and blood samples of SLE patients. While only aminority of bacterial taxa are specifically coated by endogenousintestinal IgA, IgA-coated bacteria in SLE patients had differentialrepresentation with recurrent taxa-specific expansion in SLEpatients. Strikingly, Prevotella copri, which has recently beenlinked to new-onset RA, was significantly over-representedamong the IgA-coated taxa only in SLE patients with high diseaseactivity, and was not detected in healthy controls.Conclusion Our studies provide the first evidence that SLE isassociated with gut microbiome dysbiosis with expansions of specific bacterial taxa that may contribute to immune dysregulation.This imbalance was more significant in patients with high diseaseactivity Characterisation of in vivo IgA-coated bacteria demonstrated that certain microbes taxa/species are preferentially recognised by the adaptive immune system of SLE patients, and thesediffer significantly from healthy adults. We are now studyingthese candidate pathobionts in longitudinal studies to addresswhether the microbiome predicts and/or tracks flares

At birth, the human immune system already contains substantial levels of polymeric IgM, that include autoantibodies to neo-epitopes on apoptotic cells (ACs) that are proposed to play homeostatic and anti-inflammatory roles. Yet the biologic origins and developmental regulation of these naturally arising antibodies remain poorly understood. Herein, we report that levels of IgM-antibodies to malondialdehyde (MDA) protein adducts, a common type of in vivo generated oxidative stress-related neoepitope, directly correlate with the relative binding of neonatal-IgM to ACs. Levels of IgM to phosphorylcholine (PC), a natural antibody prevalent in adults, were relatively scant in cord blood, while there was significantly greater relative representation of IgM anti-MDA antibodies in newborns compared to adults. To investigate the potential interrelationships between neonatal IgM with pathogenic IgG-autoantibodies, we studied 103 newborns born to autoimmune mothers with IgG anti-Ro (i.e., 70 with neonatal lupus and 33 without neonatal lupus). In these subjects the mean levels of IgM anti-Ro60 were significantly higher than in the newborns from non-autoimmune mothers. In contrast, levels of IgM anti-MDA in IgG anti-Ro exposed neonates were significantly lower than in neonates from non-autoimmune mothers. The presence or absence of neonatal lupus did not appear to influence the total levels of IgM in the anti-Ro exposed newborns. Taken together, our studies provide evidence that the immune development of the natural IgM-repertoire may be affected, and become imprinted by, the transfer of maternal IgG into the fetus.

BACKGROUND: Rheumatic heart disease (RHD) and HIV are prevalent diseases in sub-Saharan Africa, but little is known about their potential interrelationships. The objective of this study was to assess the prevalence of protective natural autoantibodies among patients with RHD in Uganda, and to determine whether the levels of these autoantibodies are affected by HIV status. METHODS: Participants were grouped according to RHD and HIV status. The three control groups (R