Niewold, Timothy B.

Abstract Background: The objective of the study was to explore disease pathways activated in the inflammatory foci of skin lesions in cutaneous lupus erythematosus (CLE) and dermatomyositis (DM). Methods: Skin biopsies acquired from active CLE and DM lesions, patient (PC) and also healthy controls (HC) were investigated. Biopsy sections were examined by a pathologist and inflammatory foci were laser micro-dissected, captured and proteins within captured tissue were detected in a hypothesis free manner by mass-spectrometry. Protein pathway analysis was performed by string-db.org platform. Findings of interest were confirmed by immunohistochemistry (IHC).Results: Proteome investigation identified interleukin (IL)-16 to be the only detectable and abundant cytokine differentially expressed in CLE compared to DM. Caspase-3, enzyme that cleaves IL-16 into its active form, was detected in low levels. Significantly higher proportion of IL-16 and Caspase-3 positive cells were identified in CLE lesions in comparison to DM, PC and HC. Interferon-regulated proteins (IRP) were abundant in both CLE and DM. Proteomic results indicate more abundant complement deposition in CLE skin lesions. Conclusions: Using hypothesis free mass-spectrometry investigation of CLE inflammatory infiltrates, we identified that IL-16 is the only detectable and highly abundant cytokine, while IRP was a common feature of both CLE and DM. IHC confirmed high expression of IL-16 and caspase-3 in CLE. Our novel molecular findings indicate that IL-16 detection could be useful in differential diagnostics between the two conditions that can display similar histopathological appearance. Potentially, IL-16 could be of interest as a future therapeutic target for CLE.

ObjectivePublished predictive models of disease outcomes in idiopathic inflammatory myopathies (IIMs) are sparse and of limited accuracy due to disease heterogeneity. Computational methods may address this heterogeneity by partitioning patients based on clinical and biological phenotype.MethodsTo identify new patient groups, we applied similarity network fusion (SNF) to clinical and biological data from 168 patients with myositis (64 adult polymyositis [PM], 65 adult dermatomyositis [DM], and 39 juvenile DM [JDM]) in the Rituximab in Myositis trial. We generated a sparse proof‐of‐concept bedside classifier using multinomial regression and identified characteristics that distinguished these groups. We conducted χ2 tests to link new patient groups with the myositis subtypes.ResultsSNF identified five patient groups in the discovery cohort that subdivided the myositis subtypes. The sparse multinomial regressor to predict patient group assignments (areas under the receiver operating characteristic curve = [0.78, 0.97]; areas under the precision‐recall curve = [0.55, 0.96]) found that autoantibody enrichment defined four of these groups: anti–Mi‐2, anti–signal recognition peptide (SRP), anti–nuclear matrix protein 2 (NXP2), and anti‐synthetase (Syn). Depletion of immunoglobulin M (IgM) defined the fifth group. Each group was associated with one subtype, with adult DM being associated with anti–Mi‐2 and anti‐Syn autoantibodies, JDM being associated with anti‐NXP2 autoantibodies, and adult PM being associated with IgM depletion and anti‐SRP autoantibodies. These associations enabled us to further resolve the current myositis subtypes.ConclusionUsing unsupervised machine learning, we identified clinically and biologically homogeneous groups of patients with IIMs, forming the basis of an integrated disease classification based on both clinical and biological phenotype, thus validating other approaches and what has been previously described.

ObjectiveType I interferon (IFN) is important to systemic lupus erythematosus (SLE) pathogenesis, but it is not clear how chronic elevations in IFN alter immune function. We compared cytokine responses after whole blood stimulation with Toll‐like receptor (TLR) agonists in high‐ and low‐IFN SLE patient subgroups.MethodsSLE patients and nonautoimmune controls were recruited, and SLE patients were categorized as either high or low IFN. Whole blood was dispensed into tubes coated with lipopolysaccharide (LPS), oligonucleotides with cytosine‐guanine repeats, Resiquimod, IFN‐α, and IFN‐α + LPS. Cytokine production in patient sera and after whole blood TLR stimulation was measured by multiplex assay, and type I IFN was assessed using a functional assay.ResultsCirculating plasmacytoid dendritic cell numbers were specifically reduced in high‐IFN SLE patients and not in low‐IFN SLE patients. In serum, we observed that the correlations between cytokines in serum differed to a much greater degree between the high‐ and low‐IFN groups (P < 0.0001) than the absolute cytokine levels differed between these same groups. In stimulated conditions, the high‐IFN patients had less cytokine production in response to TLR ligation than the low‐IFN SLE patients. LPS produced the most diverse response, and a number of interactions between type I IFN and LPS were observed.ConclusionWe find striking differences in resting and stimulated cytokine patterns in high‐ vs. low‐IFN SLE patients, which supports the biological importance of these patient subsets. These data could inform personalized treatment approaches and the pathogenesis of SLE flare following infection.

ObjectiveOur aim was to identify cytokines and chemokines in patients with adult dermatomyositis (DM) and juvenile dermatomyositis (JDM) that predict changes in disease activity.MethodsMultiplexed immunoassays (Meso Scale Discovery) enabled simultaneous measurement of interferon (IFN)‐regulated chemokines and other pro‐ and anti‐inflammatory cytokines specific to differentiation of specific T‐cell and innate pathways. Cytokine scores were computed for IFNCK (IP‐10, MCP‐1), Th1 (IFNɣ, TNFα, and IL2), Th2 (IL4, IL10, IL12, and IL 13), Th17 (IL6, IL17, IL1β), macrophage (MIP‐1α, MIP‐1β, IL8), and regulatory (IL10, TNFα) factors. Spearman correlation and mixed models were used to examine whether cytokines at a previous visit predict change in disease activity at the next visit.ResultsThe study included 36 patients (16 DM and 20 JDM) with at least two visits (87 patient intervals between two visits). Mean age (SD) at inclusion was 56.9 (18.4) years for DM and 10.8 (6.6) years in JDM, 67% of patients were female, 89% Caucasian. The mean (SD) physician global, muscle and extra‐muscular disease activity Visual Analog Scale scores at inclusion were 41 (26), 36 (30), and 34 (21) mm, respectively. The change in IFN score from one visit to the next was associated with the change in physician global (P = 0.010) and extramuscular (P < 0.001) disease activity scores. Preliminary results revealed significant correlations of previous IFNCK score and IL‐6 with subsequent disease activity measures, but after adjustment for multiple visits per patient, these associations did not reach statistical significance.ConclusionThere is a potential relationship between IFNCK and other cytokine scores seen in adult and juvenile DM with future disease states.

Objective The type I interferon pathway is activated in many patients with systemic lupus erythematosus (SLE), and anti-double-stranded DNA (dsDNA) and anti-RNA binding protein autoantibodies are correlated with high interferon-α (IFNα) activity. We studied whether antiphospholipid (APL) antibodies, which should not stimulate Toll-like receptors, are also associated with high levels of IFNα activity. Methods Serum IFNα activity was measured in patients with SLE using the WISH cell bioassay. IgG APL, anti-RBP and anti-dsDNA antibodies were measured in the clinical laboratory, and standard clinical cut-offs were used to define the positive results. Results High IFNα activity was associated with anti-RBP and anti-dsDNA antibodies in all three ancestral backgrounds. Strikingly, African-American subjects with a positive APL antibody test had higher IFNα activity than those without IgG APL antibodies. This was not shared with other ancestral backgrounds. This finding was independent of other autoantibody profiles, and clinical features did not differ between IgG APL antibody positive versus negative African-American patients. Conclusion The difference in association between IFNα activity and IgG APL status between ancestral backgrounds supports differences in molecular pathogenesis. This may suggest B cell hyperactivity in the setting of type I IFN in African-Americans and could suggest ways to individualise therapy.

Objective. To investigate the role of whole blood viscosity in digital ulcer (DU) development in patients with diffuse and limited Systemic sclerosis.Methods. A convenience sample of patients with Systemic sclerosis (SSc) was selected from the adult Rheumatology clinic at the University of Chicago. The study group consisted of patients with SSc (with ulcers present, a history of ulcers, and no ulcers); the control group consisted of matched healthy Rheumatology clinic staff. WBV was measured using a scanning capillary viscometer at different shear rates (1–1000 1/s).Results. Whole blood viscosity as measured by a scanning capillary viscometer was increased in patients with SSc compared to healthy controls (p<0.0001). Additionally, patients with present DU had significantly higher whole blood viscosity when compared to patients with a history of DU and patients with no history of DU (p<0.0001). These findings were most pronounced at lower shear rates between 1 and 10 1/s.Conclusion. Whole blood viscosity might be a contributing factor in DU development in patients with SSc. Further studies with larger patient cohorts are required to fully evaluate how increased WBV contributes to the development of DU and whether the currently available treatment options improve the microcirculation by influencing WBV.

ObjectiveWe describe a 38‐year‐old woman who presented with a history of inflammatory arthritis, rash, and daily fevers. She was noted to have chronic parvovirus infection with persistently detectable viral titers and a novel mutation in the ELANE gene. ELANE encodes neutrophil elastase, a neutrophil serine protease with important antimicrobial effects, and is found as part of neutrophil extracellular trap (NET) complexes. Pathogenic ELANE mutations have been identified in forms of hereditary neutropenia. However, our patient never had neutropenia. Because of the striking clinical scenario, we investigated this mutation functionally.MethodsNET formation by neutrophils was assessed by scanning electron microscopy. Neutrophil activation and neutrophil elastase production were evaluated by flow cytometry and fluorescent substrate–based functional assay, respectively. A multiplex assay was used to quantitate neutrophil inflammatory cytokine production. PyMOL software was used to generate 3‐dimensional models of mutant elastase.ResultsActivated neutrophils from the patient demonstrated a significantly decreased ability to form NETs on scanning electron microscopy, as well as quantitative defects in neutrophil activation and neutrophil elastase activity. The patient's neutrophils showed altered levels of interleukin‐12 (IL‐12) and IL‐8, which are key cytokines for antiviral immunity and neutrophil chemotaxis. Three‐dimensional mapping revealed that the mutation could alter protein folding and surface charge distribution, potentially perturbing protein trafficking. Thus, the mutation could affect neutrophil function by decreasing NETosis and altering key antiviral activities of neutrophils.ConclusionThis is the first report of a human pathogenic ELANE mutation associated with a defect in NETosis and a distinct syndrome of recurrent viral infection and chronic inflammation.

ObjectiveSystemic lupus erythematosus (SLE) is frequently characterized by activation of the type I interferon (IFN) pathway. We previously observed that a missense single‐nucleotide polymorphism (rs1049564) in the purine nucleoside phosphorylase (PNP) gene was associated with high levels of IFN in SLE. PNP is a key enzyme involved in purine metabolism. In this study, we performed functional follow‐up of this polymorphism in human cells.MethodsType I IFN was measured in patient sera, using a reporter cell assay. Structural modeling of the PNP variant was performed using PyMOL software. PNP messenger RNA (mRNA) and protein levels and type I IFN–induced gene expression were measured in lymphoblastoid cell lines with known PNP rs1049564 genotypes. The cell cycle was assayed using flow cytometry.ResultsStructural modeling indicated no major disruption in folding related to rs1049564. We observed that homozygous rs1049564 TT lymphoblastoid cells had decreased PNP mRNA expression and protein levels, and that cells with the TT genotype had reduced PNP enzymatic activity even when the amount of PNP was controlled. Cells with the TT genotype had a 2‐fold increase in S‐phase block as compared with cells with the homozygous CC phenotype. The S‐phase block could be pharmacologically reversed with hypoxanthine and adenosine, supporting the notion that relative PNP deficiency is the cause of the S‐phase block. Type I IFN–induced transcripts were increased in a dose‐response manner related to the rs1049564 T allele, at both baseline and after type I IFN stimulation.ConclusionThe PNP rs1049564 T allele is a loss‐of‐function variant that induces S‐phase block and IFN pathway activation in lymphocytes. The S‐phase block could be rescued in our in vitro experiments, suggesting the potential for personalized treatment.