High Systemic Type I Interferon Activity Is Associated With Active Class III/IV Lupus Nephritis.

OBJECTIVE: Previous studies suggest a link between high serum type I interferon (IFN) and lupus nephritis (LN). We determined whether serum IFN activity is associated with subtypes of LN and studied renal tissues and cells to understand the effect of IFN in LN. METHODS: Two hundred and twenty-one patients with systemic lupus erythematosus were studied. Serum IFN activity was measured by WISH bioassay. mRNA in situ hybridization was used in renal tissue to measure expression of the representative IFN-induced gene, IFN-induced protein with tetratricopeptide repeats-1 (IFIT1), and the plasmacytoid dendritic cell (pDC) marker gene C-type lectin domain family-4 member C (CLEC4C). Podocyte cell line gene expression was measured by real-time PCR. RESULTS: Class III/IV LN prevalence was significantly increased in patients with high serum IFN compared with those with low IFN (odds ratio 5.40, P = 0.009). In multivariate regression models, type I IFN was a stronger predictor of class III/IV LN than complement C3 or anti-dsDNA antibody, and could account for the association of these variables with LN. IFIT1 expression was increased in all classes of LN, but most in the glomerular areas of active class III/IV LN kidneys. IFIT1 expression was not closely colocalized with pDCs. IFN directly activated podocyte cell lines to induce chemokines and proapoptotic molecules. CONCLUSION: Systemic high IFN is involved in the pathogenesis of severe LN. We did not find colocalization of pDCs with IFN signature in renal tissue, and instead observed the greatest intensity of the IFN signature in glomerular areas, which could suggest a blood source of IFN.

Single-cell expression quantitative trait loci (eQTL) analysis of SLE-risk loci in lupus patient monocytes.

BACKGROUND: We performed expression quantitative trait locus (eQTL) analysis in single classical (CL) and non-classical (NCL) monocytes from patients with systemic lupus erythematosus (SLE) to quantify the impact of well-established genetic risk alleles on transcription at single-cell resolution. METHODS: Single-cell gene expression was quantified using qPCR in purified monocyte subpopulations (CD14++CD16- CL and CD14dimCD16+ NCL) from SLE patients. Novel analysis methods were used to control for the within-person correlations observed, and eQTLs were compared between cell types and risk alleles. RESULTS: The SLE-risk alleles demonstrated significantly more eQTLs in NCLs as compared to CLs (p = 0.0004). There were 18 eQTLs exclusive to NCL cells, 5 eQTLs exclusive to CL cells, and only one shared eQTL, supporting large differences in the impact of the risk alleles between these monocyte subsets. The SPP1 and TNFAIP3 loci were associated with the greatest number of transcripts. Patterns of shared influence in which different SNPs impacted the same transcript also differed between monocyte subsets, with greater evidence for synergy in NCL cells. IRF1 expression demonstrated an on/off pattern, in which expression was zero in all of the monocytes studied from some individuals, and this pattern was associated with a number of SLE risk alleles. We observed corroborating evidence of this IRF1 expression pattern in public data sets. CONCLUSIONS: We document multiple SLE-risk allele eQTLs in single monocytes which differ greatly between CL and NCL subsets. These data support the importance of the SPP1 and TNFAIP3 risk variants and the IRF1 transcript in SLE patient monocyte function.

Distinct Single Cell Gene Expression in Peripheral Blood Monocytes Correlates With Tumor Necrosis Factor Inhibitor Treatment Response Groups Defined by Type I Interferon in Rheumatoid Arthritis.

Previously, we demonstrated in test and validation cohorts that type I IFN (T1IFN) activity can predict non-response to tumor necrosis factor inhibitors (TNFi) in rheumatoid arthritis (RA). In this study, we examine the biology of non-classical and classical monocytes from RA patients defined by their pre-biologic treatment T1IFN activity. We compared single cell gene expression in purified classical (CL, n = 342) and non-classical (NC, n = 359) monocytes. In our previous work, RA patients who had either high IFNß/α activity (>1.3) or undetectable T1IFN were likely to have EULAR non-response to TNFi. In this study comparisons were made among patients grouped according to their pre-biologic treatment T1IFN activity as clinically relevant: "T1IFN undetectable (T1IFN ND) or IFNß/α >1.3" (n = 9) and "T1IFN detectable but IFNß/α ≤ 1.3" (n = 6). In addition, comparisons were made among patients grouped according to their T1IFN activity itself: "T1IFN ND," "T1IFN detected and IFNß/α ≤ 1.3," and "IFNß/α >1.3." Major differences in gene expression were apparent in principal component and unsupervised cluster analyses. CL monocytes from the T1IFN ND or IFNß/α >1.3 group were unlikely to express JAK1 and IFI27 (p < 0.0001 and p 0.0005, respectively). In NC monocytes from the same group, expression of IFNAR1, IRF1, TNFA, TLR4 (p ≤ 0.0001 for each) and others was enriched. Interestingly, JAK1 expression was absent in CL and NC monocytes from nine patients. This pattern most strongly associated with the IFNß/α>1.3 group. Differences in gene expression in monocytes among the groups suggest differential IFN pathway activation in RA patients who are either likely to respond or to have no response to TNFi. Additional transcripts enriched in NC cells of those in the T1IFN ND and IFNß/α >1.3 groups included MYD88, CD86, IRF1, and IL8. This work could suggest key pathways active in biologically defined groups of patients, and potential therapeutic strategies for those patients unlikely to respond to TNFi.

Methylarginine metabolites are associated with attenuated muscle protein synthesis in cancer-associated muscle wasting.

Cancer cachexia is characterized by reductions in peripheral lean muscle mass. Prior studies have primarily focused on increased protein breakdown as the driver of cancer-associated muscle wasting. Therapeutic interventions targeting catabolic pathways have, however, largely failed to preserve muscle mass in cachexia, suggesting that other mechanisms might be involved. In pursuit of novel pathways, we used untargeted metabolomics to search for metabolite signatures that may be linked with muscle atrophy. We injected 7-week-old C57/BL6 mice with LLC1 tumor cells or vehicle. After 21 days, tumor-bearing mice exhibited reduced body and muscle mass and impaired grip strength compared with controls, which was accompanied by lower synthesis rates of mixed muscle protein and the myofibrillar and sarcoplasmic muscle fractions. Reductions in protein synthesis were accompanied by mitochondrial enlargement and reduced coupling efficiency in tumor-bearing mice. To generate mechanistic insights into impaired protein synthesis, we performed untargeted metabolomic analyses of plasma and muscle and found increased concentrations of two methylarginines, asymmetric dimethylarginine (ADMA) and NG-monomethyl-l-arginine, in tumor-bearing mice compared with control mice. Compared with healthy controls, human cancer patients were also found to have higher levels of ADMA in the skeletal muscle. Treatment of C2C12 myotubes with ADMA impaired protein synthesis and reduced mitochondrial protein quality. These results suggest that increased levels of ADMA and mitochondrial changes may contribute to impaired muscle protein synthesis in cancer cachexia and could point to novel therapeutic targets by which to mitigate cancer cachexia.

Novel genetic associations with interferon in systemic lupus erythematosus identified by replication and fine-mapping of trait-stratified genome-wide screen.

BACKGROUND/PURPOSE: High serum interferon alpha (IFN-α) is an important heritable phenotype in systemic lupus erythematosus (SLE) which is involved in primary disease pathogenesis. High vs. low levels of IFN-α are associated with disease severity and account for some of the biological heterogeneity between SLE patients. The aim of the study was to replicate and fine-map previously detected genetic associations with serum IFN-α in SLE. METHODS: We previously undertook a case-case genome-wide association study of SLE patients stratified by ancestry and extremes of phenotype in serum IFN-α. Single nucleotide polymorphisms (SNPs) in seven loci identified in this screen were selected for follow up in a large independent cohort of 1370 SLE patients (703 European-ancestry, 432 African ancestry, and 235 Amerindian ancestry). Each ancestral background was analyzed separately, and ancestry-informative markers were used to control for ancestry and admixture. RESULTS: We find a rare haplotype spanning the promoter region of EFNA5 that is strongly associated with serum IFN-α in both African-American and European-American SLE patients (OR = 3.0, p = 3.7 × 10-6). We also find SNPs in the PPM1H, PTPRM, and NRGN regions associated with IFN-α levels in European-American, Amerindian, and African-American SLE patients respectively. Many of these associations are within regulatory regions of the gene, suggesting an impact on transcription. CONCLUSION: This study demonstrates the power of molecular sub-phenotypes to reveal genetic factors involved in complex autoimmune disease. The distinct associations observed in different ancestral backgrounds emphasize the heterogeneity of molecular pathogenesis in SLE.

Impaired cardiac performance, protein synthesis, and mitochondrial function in tumor-bearing mice.

BACKGROUND: To understand the underlying mechanisms of cardiac dysfunction in cancer, we examined cardiac function, protein synthesis, mitochondrial function and gene expression in a model of heart failure in mice injected with Lewis lung carcinoma (LLC1) cells. EXPERIMENTAL DESIGN: Seven week-old C57BL/J6 male and female mice were injected with LLC1 cells or vehicle. Cardiac ejection fraction, ventricular wall and septal thickness were reduced in male, but not female, tumor-bearing mice compared to vehicle-injected control mice. Cardiac protein synthesis was reduced in tumor-bearing male mice compared to control mice (p = 0.025). Aspect ratio and form factor of cardiac mitochondria from the tumor-bearing mice were increased compared control mice (p = 0.042 and p = 0.0032, respectively) indicating a more fused mitochondrial network in the hearts of tumor-bearing mice. In cultured cardiomyocytes maximal oxygen consumption and mitochondrial reserve capacity were reduced in cells exposed to tumor cell-conditioned medium compared to non-conditioned medium (p = 0.0059, p = 0.0010). Whole transcriptome sequencing of cardiac ventricular muscle from tumor-bearing vs. control mice showed altered expression of 1648 RNA transcripts with a false discovery rate of less than 0.05. Of these, 54 RNA transcripts were reduced ≤ 0.5 fold, and 3 RNA transcripts were increased by ≥1.5-fold in tumor-bearing mouse heart compared to control. Notably, the expression of mRNAs for apelin (Apln), the apelin receptor (Aplnr), the N-myc proto-oncogene, early growth protein (Egr1), and the transcription factor Sox9 were reduced by >50%, whereas the mRNA for growth arrest and DNA-damage-inducible, beta (Gadd45b) is increased >2-fold, in ventricular tissue from tumor-bearing mice compared to control mice. CONCLUSIONS: Lung tumor cells induce heart failure in male mice in association with reduced protein synthesis, mitochondrial function, and the expression of the mRNAs for inotropic and growth factors. These data provide new mechanistic insights into cancer-associated heart failure that may help unlock treatment options for this condition.

Type I Interferon Predicts an Alternate Immune System Phenotype in Systemic Lupus Erythematosus.

OBJECTIVE: Type 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. METHODS: SLE 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. RESULTS: Circulating 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. CONCLUSION: We 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.

Lung tumor cells inhibit bone mineralization and osteoblast activity.

Patients with non-small cell lung cancer (NSLC) often develop skeletal complications and fractures. To understand mechanisms of bone loss, we developed a murine model of non-metastatic NSLC. Decreased bone mineral density, trabecular thickness and mineralization, without an increase in bone resorption, were observed in vivo in mice injected with Lewis lung adenocarcinoma (LLC1) cells in the absence of tumor cell metastases. A decrease in trabecular bone mineral density was observed in mice injected with cell-free LLC1 CM. Plasma osteoblast biomarkers and PTH-related peptide (PTHrP) were reduced, and parathyroid hormone (PTH), 1,25-dihydroxyvitamin D, calcium and phosphate concentrations were normal in tumor-bearing mice. LLC1 cell conditioned medium (CM) inhibited alkaline phosphatase activity, osteoblast mineralization, and expression of Alpl and Ocn/Bglap mRNA in MC3T3 osteoblast cultures, whereas non-CM or CM from NIH/3T3 fibroblasts did not induce similar changes. LLC1 CM reduced Wnt3a-stimulated Tcf/Lef reporter plasmid activity and Wnt5A, Tcf1 and Lef1 mRNA expression in MC3T3 cells. Although concentrations of the Wnt inhibitor, DKK2, were increased in LLC1 CM compared to non-CM, depletion of DKK2 from LLC1 CM did not completely restore Wnt3a activity in MC3T3 cultures, and recombinant DKK2 failed to inhibit osteoblast mineralization. The data indicate that in a model of lung adenocarcinoma without bone metastases, tumor cells elaborate a secreted factor(s) that reduces bone mass, bone formation and osteoblast Wnt signaling without increases in bone resorption or calcium-regulating hormone concentrations. The factor(s) mediating this inhibition of osteoblast mineralization require further characterization.

Single-cell gene expression patterns in lupus monocytes independently indicate disease activity, interferon and therapy.

OBJECTIVES: Important findings can be masked in gene expression studies of mixed cell populations. We examined single-cell gene expression in SLE patient monocytes in the context of clinical and immunological features. METHODS: Monocytes were purified from patients with SLE and controls, and individually isolated for single-cell gene expression measurement. A panel of monocyte-related transcripts were measured in individual classical (CL) and non-classical (NCL) monocytes. RESULTS: Analyses of both CL and NCL monocytes demonstrated that many genes had a lower expression rate in SLE monocytes than in controls. Unsupervised hierarchical clustering of the CL and NCL data sets demonstrated independent clusters of cells from the patients with SLE that were related to disease activity, type I interferon (IFN) and medication use. Thus, each of these factors exerted a different impact on monocyte gene expression that could be identified separately, and a number of genes correlated uniquely with disease activity. We found within-cell correlations between genes directly induced by type I IFN-induced and other non-IFN-induced genes, suggesting the downstream biological effects of type I IFN in individual human SLE monocytes which differed between CLs and NCLs. CONCLUSIONS: In summary, single-cell gene expression in monocytes was associated with a wide range of clinical and biological features in SLE, providing much greater detail and insight into the cellular biology underlying the disease than previous mixed-cell population studies.

Brief Report: A Novel ELANE Mutation Associated With Inflammatory Arthritis, Defective NETosis, and Recurrent Parvovirus Infection.

OBJECTIVE: We 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. METHODS: NET 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. RESULTS: Activated 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. CONCLUSION: This 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.

Lupus-Associated Functional Polymorphism in PNP Causes Cell Cycle Abnormalities and Interferon Pathway Activation in Human Immune Cells.

OBJECTIVE: Systemic 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. METHODS: Type 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. RESULTS: Structural 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. CONCLUSION: The 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.

CD11b activation suppresses TLR-dependent inflammation and autoimmunity in systemic lupus erythematosus.

Genetic variations in the ITGAM gene (encoding CD11b) strongly associate with risk for systemic lupus erythematosus (SLE). Here we have shown that 3 nonsynonymous ITGAM variants that produce defective CD11b associate with elevated levels of type I interferon (IFN-I) in lupus, suggesting a direct link between reduced CD11b activity and the chronically increased inflammatory status in patients. Treatment with the small-molecule CD11b agonist LA1 led to partial integrin activation, reduced IFN-I responses in WT but not CD11b-deficient mice, and protected lupus-prone MRL/Lpr mice from end-organ injury. CD11b activation reduced TLR-dependent proinflammatory signaling in leukocytes and suppressed IFN-I signaling via an AKT/FOXO3/IFN regulatory factor 3/7 pathway. TLR-stimulated macrophages from CD11B SNP carriers showed increased basal expression of IFN regulatory factor 7 (IRF7) and IFN-ß, as well as increased nuclear exclusion of FOXO3, which was suppressed by LA1-dependent activation of CD11b. This suggests that pharmacologic activation of CD11b could be a potential mechanism for developing SLE therapeutics.

Increased pretreatment serum IFN-ß/α ratio predicts non-response to tumour necrosis factor α inhibition in rheumatoid arthritis.

OBJECTIVE: Studies suggest that circulating type I interferon (IFN) may predict response to biological agents in rheumatoid arthritis (RA). Prediction of response prior to initiating therapy would represent a major advancement. METHODS: We studied sera from a test set of 32 patients with RA from the Auto-immune Biomarkers Collaborative Network Consortium and a validation set of 92 patients with RA from the Treatment Efficacy and Toxicity in Rheumatoid Arthritis Database and Repository registry. The test set included those with good response or no response to tumour necrosis factor (TNF) inhibitors at 14 weeks by European League Against Rheumatism criteria. The validation set included subjects with good, moderate or no response at 12 weeks. Total serum type I IFN activity, IFN-α and IFN-ß activity were measured using a functional reporter cell assay. RESULTS: In the test set, an increased ratio of IFN-ß to IFN-α (IFN-ß/α activity ratio) in pretreatment serum associated with lack of response to TNF inhibition (p=0.013). Anti-cyclic citrullinated peptide antibody titre and class of TNF inhibitor did not influence this relationship. A receiver-operator curve supported a ratio of 1.3 as the optimal cut-off. In the validation set, subjects with an IFN-ß/α activity ratio >1.3 were significantly more likely to have non-response than good response (OR=6.67, p=0.018). The test had 77% specificity and 45% sensitivity for prediction of non-response compared with moderate or good response. Meta-analysis of test and validation sets confirmed strong predictive capacity of IFN-ß/α activity ratio (p=0.005). CONCLUSIONS: Increased pretreatment serum IFN-ß/α ratio strongly associated with non-response to TNF inhibition. This study supports further investigation of serum type I IFN in predicting outcome of TNF inhibition in RA.