Mycophenolate mofetil withdrawal in patients with systemic lupus erythematosus: a multicentre, open-label, randomised controlled trial.

BACKGROUND: Mycophenolate mofetil is an immunosuppressant commonly used to treat systemic lupus erythematosus (SLE) and lupus nephritis. It is a known teratogen associated with significant toxicities, including an increased risk of infections and malignancies. Mycophenolate mofetil withdrawal is desirable once disease quiescence is reached, but the timing of when to do so and whether it provides a benefit has not been well-studied. We aimed to determine the effects of mycophenolate mofetil withdrawal on the risk of clinically significant disease reactivation in patients with quiescent SLE on long-term mycophenolate mofetil therapy. METHODS: This multicenter, open-label, randomised trial was conducted in 19 centres in the USA. Eligible patients were aged between 18 and 70 years old, met the American College of Rheumatology (ACR) 1997 SLE criteria, and had a clinical SLEDAI score of less than 4 at screening. Mycophenolate mofetil therapy was required to be stable or decreasing for 2 years or more if initiated for renal indications, or for 1 year or more for non-renal indications. Participants were randomly allocated in a 1:1 ratio to a withdrawal group, who tapered off mycophenolate mofetil over 12 weeks, or a maintenance group who maintained their baseline dose (1-3g per day) for 60 weeks. Adaptive random allocation ensured groups were balanced for study site, renal versus non-renal disease, and baseline mycophenolate mofetil dose (≥2 g per day vs <2 g per day). Clinically significant disease reactivation by week 60 following random allocation, requiring increased doses or new immunosuppressive therapy was the primary endpoint, in the modified intention-to-treat population (all randomly allocated participants who began study-provided mycophenolate mofetil). Non-inferiority was evaluated using an estimation-based approach. The trial was registered at ClinicalTrials.gov (NCT01946880) and is completed. FINDINGS: Between Nov 6, 2013, and April 27, 2018, 123 participants were screened, of whom 102 were randomly allocated to the maintenance group (n=50) or the withdrawal group (n=52). Of the 100 participants included in the modified intention-to-treat analysis (49 maintenance, 51 withdrawal), 84 (84%) were women, 16 (16%) were men, 40 (40%) were White, 41 (41%) were Black, and 76 (76%) had a history of lupus nephritis. The average age was 42 (SD 12·7). By week 60, nine (18%) of 51 participants in the withdrawal group had clinically significant disease reactivation, compared to five (10%) of 49 participants in the maintenance group. The risk of clinically significant disease reactivation was 11% (95% CI 5-24) in the maintenance group and 18% (10-32) in the withdrawal group. The estimated increase in the risk of clinically significant disease reactivation with mycophenolate mofetil withdrawal was 7% (one-sided upper 85% confidence limit 15%). Similar rates of adverse events were observed in the maintenance group (45 [90%] of 50 participants) and the withdrawal group (46 [88%] of 52 participants). Infections were more frequent in the mycophenolate mofetil maintenance group (32 [64%]) compared with the withdrawal group (24 [46%]). INTERPRETATIONS: Mycophenolate mofetil withdrawal is not significantly inferior to mycophenolate mofetil maintenance. Estimates for the rates of disease reactivation and increases in risk with withdrawal can assist clinicians in making informed decisions on withdrawing mycophenolate mofetil in patients with stable SLE. FUNDING: The National Institute of Allergy and Infectious Diseases and the National Institute of Arthritis and Musculoskeletal and Skin Diseases.

A single center pilot study: assessing resident needs and faculty perceptions to improve training in rheumatology.

BACKGROUND: Internal medicine (IM) residents lack confidence in rheumatology. Due to the wide variety of topics in rheumatology, identifying the most important subjects to learn during training is vital to create future interventions to increase confidence and knowledge. The preferred teaching modality for both attendings/fellows and residents is not known. METHODS: An electronic survey was distributed to all IM residents, rheumatology fellows, and rheumatology faculty at the University of Chicago during the 2020-2021 academic year. Residents reported self-confidence levels on 10 rheumatology topics, while rheumatology attendings/fellows were asked to rank these from most to least important to learn during IM residency. All groups were asked preferred teaching modality. RESULTS: Median confidence level [interquartile range] among residents for caring for patients with rheumatological conditions was 6 [3.6-7.5] for inpatient and 5 [3.7-6.5] for outpatient settings (10 being very confident). Attendings and fellows identified the most important topics to learn during the rheumatology rotation as ordering and interpreting autoimmune serologies and musculoskeletal exam. Both attendings/fellows and residents preferred bedside teaching in the inpatient setting and case-based learning in the outpatient setting. CONCLUSIONS: While some disease-specific topics such as autoimmune serologies were identified as important rheumatology topics for IM residents to learn, more practical topics like musculoskeletal exam skills were also deemed important. This highlights the need for comprehensive interventions that focus on more than standardized exam topics alone to improve rheumatology confidence in IM residents. There are different preferences of teaching styles in various clinical settings.

Specific in situ inflammatory states associate with progression to renal failure in lupus nephritis.

BACKGROUNDIn human lupus nephritis (LN), tubulointerstitial inflammation (TII) on biopsy predicts progression to end-stage renal disease (ESRD). However, only about half of patients with moderate-to-severe TII develop ESRD. We hypothesized that this heterogeneity in outcome reflects different underlying inflammatory states. Therefore, we interrogated renal biopsies from LN longitudinal and cross-sectional cohorts.METHODSData were acquired using conventional and highly multiplexed confocal microscopy. To accurately segment cells across whole biopsies, and to understand their spatial relationships, we developed computational pipelines by training and implementing several deep-learning models and other computer vision techniques.RESULTSHigh B cell densities were associated with protection from ESRD. In contrast, high densities of CD8+, γδ, and other CD4-CD8- T cells were associated with both acute renal failure and progression to ESRD. B cells were often organized into large periglomerular neighborhoods with Tfh cells, while CD4- T cells formed small neighborhoods in the tubulointerstitium, with frequency that predicted progression to ESRD.CONCLUSIONThese data reveal that specific in situ inflammatory states are associated with refractory and progressive renal disease.FUNDINGThis study was funded by the NIH Autoimmunity Centers of Excellence (AI082724), Department of Defense (LRI180083), Alliance for Lupus Research, and NIH awards (S10-OD025081, S10-RR021039, and P30-CA14599).

Innate-like self-reactive B cells infiltrate human renal allografts during transplant rejection.

Intrarenal B cells in human renal allografts indicate transplant recipients with a poor prognosis, but how these cells contribute to rejection is unclear. Here we show using single-cell RNA sequencing that intrarenal class-switched B cells have an innate cell transcriptional state resembling mouse peritoneal B1 or B-innate (Bin) cells. Antibodies generated by Bin cells do not bind donor-specific antigens nor are they enriched for reactivity to ubiquitously expressed self-antigens. Rather, Bin cells frequently express antibodies reactive with either renal-specific or inflammation-associated antigens. Furthermore, local antigens can drive Bin cell proliferation and differentiation into plasma cells expressing self-reactive antibodies. These data show a mechanism of human inflammation in which a breach in organ-restricted tolerance by infiltrating innate-like B cells drives local tissue destruction.

The impact of education and clinical decision support on the quality of positive antinuclear antibody referrals.

To implement and evaluate an intervention using education and clinical decision support (CDS) to improve the quality of positive ANA referrals. We retrospectively reviewed "positive ANA" referrals from April 2017 to May 2019. Demographic data and referring provider's location were recorded. Final diagnoses were categorized into two groups: rheumatic disease (RD) or no RD. We compared pre- and post-intervention groups for each type of referral. The positive predictive value (PPV) of an ANA referral leading to an RD for each referral group was calculated. Our intervention consisted of an educational poster and CDS which included a hard-stop prompt embedded into the electronic ANA order. All internal subgroups received CDS; only the main campus primary care providers (IPCP) received the educational poster. The external (EXT) referral subgroup did not receive either intervention. We found a significant increase in the number of RDs diagnosed post-intervention (p = 0.007). The PPV for all referrals increased from 16% to 26% during this project. All groups demonstrated improvement in PPV except the EXT group, which showed no change. Subgroups which demonstrated significant increase in the diagnosis of RD included total internal (p = 0.0005), internal PCP (p = 0.002), and affiliated primary care providers (p = 0.0002). The IPCP subgroup additionally received the educational intervention and did not demonstrate significant improvement. Implementing an intervention with a CDS component helps improve the quality of positive ANA referrals to rheumatology. Key Points • Clinical decision support improves the quality of positive ANA referrals. • Incorporating clinical decision support within the ANA order of an EHR is an effective way to deliver information to impact ordering at the "point of care."

Cellular aspects of the pathogenesis of lupus nephritis.

PURPOSE OF REVIEW: Lupus nephritis is a common severe manifestation of systemic lupus erythematosus. Despite recent advances in therapeutics and understanding of its pathogenesis, there are still substantial unmet needs. This review discusses recent discoveries in these areas, especially the role of tubulointerstitial inflammation (TII) in lupus nephritis. RECENT FINDINGS: Non-white ethnicity is still a major risk and poor prognostic factor in lupus nephritis. TII and fibrosis have been found to be associated with worse renal outcome but the current lupus nephritis treatment guidelines and trials are based on the degree of glomerular inflammation. In combination with mycophenolate mofetil, a B-cell-targeted therapy (belimumab) and a calcineurin inhibitor (voclosporin) have shown efficacy in recent lupus nephritis trials. However, response rates have been modest. While lupus glomerulonephritis results from immune complex deposition derived from systemic autoantibodies, TII arises from complex processes associated with in situ adaptive cell networks. These include local antibody production, and cognate or antigen-induced interactions between T follicular helper cells, and likely other T-cell populations, with antigen presenting cells including B cells, myeloid dendritic cells and plasmacytoid dendritic cells. SUMMARY: Better understanding of the pathogenesis of TII will identify novel therapeutic targets predicted to improve outcomes in our patients with lupus nephritis.

Bcl-2 as a Therapeutic Target in Human Tubulointerstitial Inflammation.

OBJECTIVE: In lupus nephritis, tubulointerstitial inflammation (TII) is associated with in situ adaptive immune cell networks that amplify local tissue damage. Since conventional therapy appears ineffective for severe TII, and these patients often progress to renal failure, understanding in situ mechanisms might reveal new therapeutic targets. This study was undertaken to assess whether dysregulated apoptotic regulators maintain local adaptive immunity and drive inflammation in TII. METHODS: This study utilized novel computational approaches that, when applied to multicolor confocal images, quantified apoptotic regulator protein expression in selected lymphocyte subsets. This approach was validated using laser-capture microdissection (LCM) coupled to quantitative polymerase chain reaction (qPCR). Furthermore, the consequences of dysregulated apoptotic mediator expression were explored in a murine model of lupus nephritis. RESULTS: Analyses of renal biopsy tissue from patients with lupus nephritis and those with mixed cellular renal allograft rejection revealed that the B cell lymphoma 2 protein (Bcl-2) was frequently expressed in infiltrating lymphocytes, whereas expression of myeloid cell leukemia 1 was low. In contrast, the reciprocal pattern of expression was observed in tonsil germinal centers. These results were consistent with RNA expression data obtained using LCM and qPCR. Bcl-2 was also highly expressed in tubulointerstitial infiltrates in (NZB × NZW)F1 (NZB/NZW) mice. Furthermore, treatment of NZB/NZW mice with ABT-199, a selective oral inhibitor of Bcl-2, prolonged survival and prevented proteinuria and development of TII in a lupus prevention model. Interestingly, glomerular immune complexes were partially ameliorated by ABT-199 treatment, and serum anti-double-stranded DNA antibody titers were unaffected. CONCLUSION: These data demonstrate that Bcl-2 is an attractive therapeutic target in patients with lupus nephritis who manifest TII.

Widely divergent transcriptional patterns between SLE patients of different ancestral backgrounds in sorted immune cell populations.

Systemic lupus erythematosus (SLE) is a complex autoimmune disease of uncertain etiology. Patients from different ancestral backgrounds demonstrate differences in clinical manifestations and autoantibody profiles. We examined genome-wide transcriptional patterns in major immune cell subsets across different ancestral backgrounds. Peripheral blood was collected from African-American (AA) and European-American (EA) SLE patients and controls. CD4 T-cells, CD8 T-cells, monocytes, and B cells were purified by flow sorting, and each cell subset from each subject was run on a genome-wide expression array. Cases were compared to controls of the same ancestral background. The overlap in differentially expressed gene (DEG) lists between different cell types from the same ancestral background was modest (<10%), and only 5-8% overlap in DEG lists was observed when comparing the same cell type between different ancestral backgrounds. IFN-stimulated gene (ISG) expression was not up-regulated synchronously in all cell types from a given patient, for example a given subject could have high ISG expression in T and B cells, but not in monocytes. AA subjects demonstrated more concordance in ISG expression between cell types from the same individual, and AA patients demonstrated significant down-regulation of metabolic gene expression which was not observed in EA patients. ISG expression was significantly decreased in B cells in patients taking immunosuppressants, while ISGs in other cell types did not differ with medication use. In conclusion, gene expression was strikingly different between immune cell subsets and between ancestral backgrounds in SLE patients. These findings emphasize the critical importance of studying multiple ancestral backgrounds and multiple cell types in gene expression studies. Ancestral backgrounds which are not studied will not benefit from personalized medicine strategies in SLE.

Vimentin is a dominant target of in situ humoral immunity in human lupus tubulointerstitial nephritis.

OBJECTIVE: In lupus nephritis (LN), severe tubulointerstitial inflammation (TII) predicts progression to renal failure. Severe TII is associated with tertiary lymphoid neogenesis and in situ antigen-driven clonal B cell selection. The autoantigen(s) driving in situ B cell selection in TII are not known. This study was undertaken to identify the dominant driving autoantigen(s). METHODS: Single CD38+ or Ki-67+ B cells were laser captured from 7 biopsy specimens that were diagnostic for LN. Eighteen clonally expanded immunoglobulin heavy- and light-chain variable region pairs were cloned and expressed as monoclonal antibodies. Seven more antibodies were cloned from flow-sorted CD38+ cells from an eighth biopsy specimen. Antigen characterization was performed using a combination of confocal microscopy, enzyme-linked immunosorbent assay, screening protoarrays, immunoprecipitation, and mass spectrometry. Serum IgG titers to the dominant antigen in 48 LN and 35 non-nephritic lupus samples were determined using purified antigen-coated arrays. Autoantigen expression on normal and LN kidney was localized by immunohistochemistry and immunofluorescence. RESULTS: Eleven of 25 antibodies reacted with cytoplasmic structures, 4 reacted with nuclei, and none reacted with double-stranded DNA. Vimentin was the only autoantigen identified by both mass spectrometry and protoarray. Ten of the 11 anticytoplasmic TII antibodies directly bound vimentin. Vimentin was highly expressed by tubulointerstitial inflammatory cells, and the TII antibodies tested preferentially bound inflamed tubulointerstitium. Finally, high titers of serum antivimentin antibodies were associated with severe TII (P = 0.0001). CONCLUSION: Vimentin, an antigenic feature of inflammation, is a dominant autoantigen targeted in situ in LN TII. This adaptive autoimmune response likely feeds forward to worsen TII and renal damage.

The emerging role of the inflammasome in kidney diseases.

PURPOSE OF REVIEW: To focus on the latest data that elucidate the role of the NLRP3 inflammasome in kidney diseases. RECENT FINDINGS: The NLRP3 inflammasome is not limited by the traditional microbial stimuli of innate immunity and its connection with autophagy, apoptosis, fibrosis, and pro-inflammatory cytokines has broader implications for a variety of kidney diseases. In a wide spectrum of glomerular and tubulointerstitial diseases, the NLRP3 inflammasome is upregulated in both classical immune cells such as infiltrating macrophages and resident dendritic cells as well as in renal tubular epithelial cells, and even podocytes. Inhibition of the NLRP3 inflammasome ameliorates renal injury in a variety of animal models. Interestingly, this extends to models of proteinuria, which suggests that the deleterious effect of albuminuria on the proximal tubular epithelium and podocytes is, in part, mediated by inflammasome activation. SUMMARY: Recent studies in animal models, and limited studies in humans, suggest a broad role for inflammasome activation in renal disease. Surprisingly, individual components of the inflammasome, independent of inflammasome activation, may also contribute to progressive renal injury. Additional, studies are needed to define the relative importance of the inflammasome in specific diseases and the therapeutic opportunities afforded by targeting the inflammasome.

Activation of the Interferon Pathway is Dependent Upon Autoantibodies in African-American SLE Patients, but Not in European-American SLE Patients.

BACKGROUND: In systemic lupus erythematosus (SLE), antibodies directed at RNA-binding proteins (anti-RBP) are associated with high serum type I interferon (IFN), which plays an important role in SLE pathogenesis. African-Americans (AA) are more likely to develop SLE, and SLE is also more severe in this population. We hypothesized that peripheral blood gene expression patterns would differ between AA and European-American (EA) SLE patients, and between those with anti-RBP antibodies and those who lack these antibodies. METHODS: Whole blood RNA from 33 female SLE patients and 16 matched female controls from AA and EA ancestral backgrounds was analyzed on Affymetrix Gene 1.0 ST gene expression arrays. Ingenuity Pathway Analysis was used to compare the top differentially expressed canonical pathways amongst the sample groups. An independent cohort of 116 SLE patients was used to replicate findings using quantitative real-time PCR (qPCR). RESULTS: Both AA and EA patients with positive anti-RBP antibodies showed over-expression of similar IFN-related canonical pathways, such as IFN Signaling (P = 1.3 × 10(-7) and 6.3 × 10(-11) in AA vs. EA respectively), Antigen Presenting Pathway (P = 1.8 × 10(-5) and 2.5 × 10(-6)), and a number of pattern recognition receptor pathways. In anti-RBP negative (RBP-) patients, EA subjects demonstrated similar IFN-related pathway activation, whereas no IFN-related pathways were detected in RBP-AA patients. qPCR validation confirmed similar results. CONCLUSION: Our data show that IFN-induced gene expression is completely dependent on the presence of autoantibodies in AA SLE patients but not in EA patients. This molecular heterogeneity suggests differences in IFN-pathway activation between ancestral backgrounds in SLE. This heterogeneity may be clinically important, as therapeutics targeting this pathway are being developed.

Interferon regulatory factor 5 in the pathogenesis of systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by multiple genetic risk factors, high levels of interferon alpha (IFN-α), and the production of autoantibodies against components of the cell nucleus. Interferon regulatory factor 5 (IRF5) is a transcription factor which induces the transcription of IFN-α and other cytokines, and genetic variants of IRF5 have been strongly linked to SLE pathogenesis. IRF5 functions downstream of Toll-like receptors and other microbial pattern-recognition receptors, and immune complexes made up of SLE-associated autoantibodies seem to function as a chronic endogenous stimulus to this pathway. In this paper, we discuss the physiologic role of IRF5 in immune defense and the ways in which IRF5 variants may contribute to the pathogenesis of human SLE. Recent data regarding the role of IRF5 in both serologic autoimmunity and the overproduction of IFN-α in human SLE are summarized. These data support a model in which SLE-risk variants of IRF5 participate in a "feed-forward" mechanism, predisposing to SLE-associated autoantibody formation, and subsequently facilitating IFN-α production downstream of Toll-like receptors stimulated by immune complexes composed of these autoantibodies.

Gene-expression-guided selection of candidate loci and molecular phenotype analyses enhance genetic discovery in systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is a highly heterogeneous autoimmune disorder characterized by differences in autoantibody profiles, serum cytokines, and clinical manifestations. We have previously conducted a case-case genome-wide association study (GWAS) of SLE patients to detect associations with autoantibody profile and serum interferon alpha (IFN-α). In this study, we used public gene expression data sets to rationally select additional single nucleotide polymorphisms (SNPs) for validation. The top 200 GWAS SNPs were searched in a database which compares genome-wide expression data to genome-wide SNP genotype data in HapMap cell lines. SNPs were chosen for validation if they were associated with differential expression of 15 or more genes at a significance of P < 9 × 10(-5). This resulted in 11 SNPs which were genotyped in 453 SLE patients and 418 matched controls. Three SNPs were associated with SLE-associated autoantibodies, and one of these SNPs was also associated with serum IFN-α (P < 4.5 × 10(-3) for all). One additional SNP was associated exclusively with serum IFN-α. Case-control analysis was insensitive to these molecular subphenotype associations. This study illustrates the use of gene expression data to rationally select candidate loci in autoimmune disease, and the utility of stratification by molecular phenotypes in the discovery of additional genetic associations in SLE.

Genetic ancestry, serum interferon-α activity, and autoantibodies in systemic lupus erythematosus.

OBJECTIVE: To investigate and refine the relationships among systemic lupus erythematosus (SLE) and related autoantibodies, interferon-α (IFN-α), and various ancestral backgrounds. METHODS: We investigated quantitatively defined genetic ancestry through principal component analysis in place of self-reported ancestry. RESULTS: African ancestry was found to be associated with presence of anti-RNP antibody (p = 0.0026), and anti-RNP was correlated with high levels of IFN-α (p = 2.8 × 10(-5)). CONCLUSION: Our data support a model in which African ancestry increases the likelihood of SLE-associated autoantibody formation, which subsequently results in higher levels of serum IFN-α.

The false negative technetium-99m-sestamibi scan in patients with primary hyperparathyroidism: correlation with clinical factors and operative findings.

False negative (FN) results limit the efficacy of technetium-99m-sestamibi scanning for parathyroid localization. We determined the incidence of FN results and attempted to correlate it with clinical and operative findings. One hundred forty-six patients underwent parathyroidectomy; 89 had primary hyperparathyroidism (76 single adenoma and 13 multiglandular disease) and underwent sestamibi scanning. The false negative rate was 22 per cent with an overall sensitivity of 77 per cent and a positive predictive value of 99 per cent. Patients with single adenomas were more likely to have a true positive scan than those with multiglandular disease [83% vs 38%; odds ratio (OR) = 7.754, 95% confidence interval (CI) = 2.184-27.524; P < or = 0.0001]. Inferior adenomas (90% vs 59%; OR = 6.261, 95% CI = 2.037-19.243; P < or = 0.0001) and larger adenomas (1422.3 +/- 1576.2 vs 474.6 +/- 193.2 g; P < or = 0.0001) were more likely to be detected by sestamibi imaging. Patients with normal preoperative calcium levels were more likely to have an FN sestamibi scan. Sestamibi parathyroid imaging is limited by a 22 per cent FN rate and is less accurate for detecting abnormal parathyroid tissue in patients with small adenomas, multiglandular disease, superior adenomas, or preoperative normocalcemia.