Systemic cytokines and GlycA discriminate disease status and predict corticosteroid response in HTLV-1-associated neuroinflammation.

BACKGROUND: HTLV-1-Associated Myelopathy/Tropical Spastic Paraparesis (HAM/TSP) is an incapacitating neuroinflammatory disorder for which no disease-modifying therapy is available, but corticosteroids provide some clinical benefit. Although HAM/TSP pathogenesis is not fully elucidated, older age, female sex and higher proviral load are established risk factors. We investigated systemic cytokines and a novel chronic inflammatory marker, GlycA, as possible biomarkers of immunopathogenesis and therapeutic response in HAM/TSP, and examined their interaction with established risk factors. PATIENTS AND METHODS: We recruited 110 People living with HTLV-1 (PLHTLV-1, 67 asymptomatic individuals and 43 HAM/TSP patients) with a total of 946 person-years of clinical follow-up. Plasma cytokine levels (IL-2, IL-4, IL-6, IL-10, IL-17A, IFN-γ, TNF) and GlycA were quantified by Cytometric Bead Array and 1NMR, respectively. Cytokine signaling and prednisolone response were validated in an independent cohort by nCounter digital transcriptomics. We used multivariable regression, machine learning algorithms and Bayesian network learning for biomarker identification. RESULTS: We found that systemic IL-6 was positively correlated with both age (r = 0.50, p < 0.001) and GlycA (r = 0.45, p = 0.00049) in asymptomatics, revealing an 'inflammaging" signature which was absent in HAM/TSP. GlycA levels were higher in women (p = 0.0069), but cytokine levels did not differ between the sexes. IFN-γ (p = 0.007) and IL-17A (p = 0.0001) levels were increased in untreated HAM/TSP Multivariable logistic regression identified IL-17A and proviral load as independent determinants of clinical status, resulting in modest accuracy of predicting HAM/TSP status (64.1%), while a machine learning-derived decision tree classified HAM/TSP patients with 90.7% accuracy. Pre-treatment GlycA and TNF levels significantly predicted clinical worsening (measured by Osame Motor Disability Scale), independent of proviral load. In addition, a poor prednisolone response was significantly correlated with higher post-treatment IFN-γ levels. Likewise, a transcriptomic IFN signaling score, significantly correlated with previously proposed HAM/TSP biomarkers (CASP5/CXCL10/FCGR1A/STAT1), was efficiently blunted by in vitro prednisolone treatment of PBMC from PLHTLV-1 and incident HAM/TSP. CONCLUSIONS: An age-related increase in systemic IL-6/GlycA levels reveals inflammaging in PLHTLV-1, in the absence of neurological disease. IFN-γ and IL-17A are biomarkers of untreated HAM/TSP, while pre-treatment GlycA and TNF predict therapeutic response to prednisolone pulse therapy, paving the way for a precision medicine approach in HAM/TSP.

Urinary Peptides as Potential Non-Invasive Biomarkers for Lupus Nephritis: Results of the Peptidu-LUP Study.

Background: Lupus nephritis (LN) is a severe manifestation of Systemic Lupus Erythematosus (SLE). The therapeutic strategy relies on kidney biopsy (KB) results. We tested whether urinary peptidome analysis could non-invasively differentiate active from non-active LN. Design: Urinary samples were collected from 93 patients (55 with active LN and 38 with non-active LN), forming a discovery (n = 42) and an independent validation (n = 51) cohort. Clinical characteristics were collected at inclusion and prospectively for 24 months. The urinary peptidome was analyzed by capillary-electrophoresis coupled to mass-spectrometry, comparing active LN to non-active LN, and assessing chronic lesions and response to therapy. The value of previously validated prognostic (CKD273) and differential diagnostic (LN172) signatures was evaluated. Results: Urinary peptides could not discriminate between active and non-active LN or predict early response to therapy. Tubulo-interstitial fibrosis was correlated to the CKD273. The LN172 score identified 92.5% of samples as LN. Few patients developed new-onset CKD. Conclusions: We validated the CKD273 and LN172 classifiers but did not identify a robust signature that could predict active LN and replace KB. The value of urinary peptidome to predict long-term CKD, or renal flares in SLE, remains to be evaluated.

Serum GlycA Level is Elevated in Active Systemic Lupus Erythematosus and Correlates to Disease Activity and Lupus Nephritis Severity.

OBJECTIVE: Reliable non-invasive biomarkers are needed to assess disease activity and prognosis in patients with systemic lupus erythematosus (SLE). Glycoprotein acetylation (GlycA), a novel biomarker for chronic inflammation, has been reported to be increased in several inflammatory diseases. We investigated the relevance of serum GlycA in SLE patients exhibiting various levels of activity and severity, especially with regards to renal involvement. METHODS: Serum GlycA was measured by nuclear magnetic resonance spectroscopy in samples from well characterized SLE patients and from both healthy controls and patients with other kidney diseases (KD). Disease activity was evaluated using the Systemic Lupus Erythematosus Activity Index 2000 (SLEDAI-2K). Renal severity was assessed by kidney biopsy. RESULTS: Serum GlycA was elevated in active (n = 105) compared to quiescent SLE patients (n = 39, p < 10-6), healthy controls (n = 20, p = 0.009) and KD controls (n = 21, p = 0.04), despite a more severely altered renal function in the latter. GlycA level was correlated to disease activity (SLEDAI-2K, ρ = 0.37, p < 10-4), Creactive protein, neutrophil count, triglyceride levels, proteinuria and inversely to serum albumin. In patients with biopsy-proven lupus nephritis (LN), GlycA levels were higher in proliferative (n = 26) than non-proliferative LN (n = 10) in univariate analysis (p = 0.04), and was shown to predict proliferative LN independently of renal parameters, immunological activity, neutrophil count and daily corticosteroid dosage by multivariate analysis (p < 5 × 10-3 for all models). In LN patients with repeated longitudinal GlycA measurement (n = 11), GlycA varied over time and seemed to peak at the time of the flare. CONCLUSIONS: GlycA, as a summary measure for different inflammatory processes, could be a valuable biomarker of disease activity in patients with SLE, and a non-invasive biomarker of pathological severity in the context of LN.

An evolutionary recent IFN/IL-6/CEBP axis is linked to monocyte expansion and tuberculosis severity in humans.

Monocyte counts are increased during human tuberculosis (TB) but it has not been determined whether Mycobacterium tuberculosis (Mtb) directly regulates myeloid commitment. We demonstrated that exposure to Mtb directs primary human CD34+ cells to differentiate into monocytes/macrophages. In vitro myeloid conversion did not require type I or type II IFN signaling. In contrast, Mtb enhanced IL-6 responses by CD34+ cell cultures and IL-6R neutralization inhibited myeloid differentiation and decreased mycobacterial growth in vitro. Integrated systems biology analysis of transcriptomic, proteomic and genomic data of large data sets of healthy controls and TB patients established the existence of a myeloid IL-6/IL6R/CEBP gene module associated with disease severity. Furthermore, genetic and functional analysis revealed the IL6/IL6R/CEBP gene module has undergone recent evolutionary selection, including Neanderthal introgression and human pathogen adaptation, connected to systemic monocyte counts. These results suggest Mtb co-opts an evolutionary recent IFN-IL6-CEBP feed-forward loop, increasing myeloid differentiation linked to severe TB in humans.

Decreased RORC expression and downstream signaling in HTLV-1-associated adult T-cell lymphoma/leukemia uncovers an antiproliferative IL17 link: A potential target for immunotherapy?

Retinoic acid-related drugs have shown promising pre-clinical activity in Adult T-cell Leukemia/Lymphoma, but RORC signaling has not been explored. Therefore, we investigated transcriptome-wide interactions of the RORC pathway in HTLV-1 and ATL, using our own and publicly available gene expression data for ATL and other leukemias. Gene expression data from ATL patients were analyzed using WGCNA to determine gene modules and their correlation to clinical and molecular data. Both PBMCs and CD4+ T-Cells exhibited decreased RORC expression in four different ATL cohorts. A small subset of RORChi ATL patients was identified with significantly lower pathognomonic CADM1 and HBZ levels but similar levels of other ATL markers (CD4/CD25/CCR4), hinting at a less aggressive ATL subtype. An age-dependent decrease in RORC expression was found in HTLV-1-infected individuals, but not in healthy controls, suggesting an early molecular event predisposing to leukemogenesis. Genes upstream of RORC signaling were members of a proliferative gene module (containing proliferation markers PCNA/Ki67), whereas downstream members clustered in an anti-proliferative gene module. IL17C transcripts showed the strongest negative correlation to PCNA in both ATL cohorts, which was replicated in two large cohorts of T- and B-cell acute lymphoid leukemia (ALL). Finally, IL17C expression in purified CD4 + CCR4 + CD26-CD7- "ATL-like" cells from HTLV-1-infected individuals and ATL patients was negatively correlated with clonality, underscoring a possible antileukemic/antiproliferative role. In conclusion, decreased RORC expression and downstream signaling might represent an early event in ATL pathogenesis. An antiproliferative IL17C/PCNA link is shared between ATL, T-ALL and B-ALL, suggesting (immuno)therapeutic benefit of boosting RORC/IL17 signaling.

GlycA, a Nuclear Magnetic Resonance Spectroscopy Measure for Protein Glycosylation, is a Viable Biomarker for Disease Activity in IBD.

BACKGROUND AND AIMS: Glycoprotein acetylation [GlycA] is a novel nuclear magnetic resonance [NMR] biomarker, measured in serum or plasma, that summarizes the signals originating from glycan groups of certain acute-phase glycoproteins. This biomarker has been shown to be robustly associated with cardiovascular and short-term all-cause mortality, and with disease severity in several inflammatory conditions. We investigated GlycA levels in a cohort of healthy individuals [HCs], patients with Crohn's disease [CD] and patients with ulcerative colitis [UC] prior to and after therapeutic control of inflammation. METHODS: Serum samples of 10 HCs, 37 CD patients and 21 UC patients before and after biologic therapy were subjected to high-throughput NMR analysis by Nightingale Health Ltd. Paired C-reactive protein [CRP] and fecal calprotectin [fCal] measurements were used to characterize baseline differences, treatment effects and post-treatment association with endoscopic response [50% SES-CD decrease at Week 24] and mucosal healing [SES-CD ≤ 2 for CD, Mayo endoscopic score ≤ 1 for UC]. RESULTS: GlycA levels were significantly higher in patients with active inflammamtory bowel disease [IBD] compared with those in healthy controls, and accurately reflected the mucosal recovery to a 'healthy' state in both CD and UC patients achieving mucosal healing. In CD patients who experienced an endoscopic response without achieving full mucosal healing, GlycA levels also decreased but did not normalize to HC levels. Overall, GlycA correlated well with CRP and fCal, and accurately tracked disease activity in CRP-negative patients [<5 mg/dL]. CONCLUSION: GlycA holds promise as a viable serological biomarker for disease activity in IBD, even in patients without elevated CRP, and should therefore be tested in large prospective cohorts.

Comprehensive Antiretroviral Restriction Factor Profiling Reveals the Evolutionary Imprint of the ex Vivo and in Vivo IFN-ß Response in HTLV-1-Associated Neuroinflammation.

HTLV-1-Associated Myelopathy (HAM/TSP) is a progressive neuroinflammatory disorder for which no disease-modifying treatment exists. Modest clinical benefit from type I interferons (IFN-α/ß) in HAM/TSP contrasts with its recently identified IFN-inducible gene signature. In addition, IFN-α treatment in vivo decreases proviral load and immune activation in HAM/TSP, whereas IFN-ß therapy decreases tax mRNA and lymphoproliferation. We hypothesize this "IFN paradox" in HAM/TSP might be explained by both cell type- and gene-specific effects of type I IFN in HTLV-1-associated pathogenesis. Therefore, we analyzed ex vivo transcriptomes of CD4+ T cells, PBMCs and whole blood in healthy controls, HTLV-1-infected individuals, and HAM/TSP patients. First, we used a targeted approach, simultaneously quantifying HTLV-1 mRNA (HBZ, Tax), proviral load and 42 host genes with known antiretroviral (anti-HIV) activity in purified CD4+ T cells. This revealed two major clusters ("antiviral/protective" vs. "proviral/deleterious"), as evidenced by significant negative (TRIM5/TRIM22/BST2) vs. positive correlation (ISG15/PAF1/CDKN1A) with HTLV-1 viral markers and clinical status. Surprisingly, we found a significant inversion of antiretroviral activity of host restriction factors, as evidenced by opposite correlation to in vivo HIV-1 vs. HTLV-1 RNA levels. The anti-HTLV-1 effect of antiviral cluster genes was significantly correlated to their adaptive chimp/human evolution score, for both Tax mRNA and PVL. Six genes of the proposed antiviral cluster underwent lentivirus-driven purifying selection during primate evolution (TRIM5/TRIM22/BST2/APOBEC3F-G-H), underscoring the cross-retroviral evolutionary imprint. Secondly, we examined the genome-wide type I IFN response in HAM/TSP patients, following short-term ex vivo culture of PBMCs with either IFN-α or IFN-ß. Microarray analysis evidenced 12 antiretroviral genes (including TRIM5α/TRIM22/BST2) were significantly up-regulated by IFN-ß, but not IFN-α, in HAM/TSP. This was paralleled by a significant decrease in lymphoproliferation by IFN-ß, but not IFN-α treatment. Finally, using published ex vivo whole blood transcriptomic data of independent cohorts, we validated the significant positive correlation between TRIM5, TRIM22, and BST2 in HTLV-1-infected individuals and HAM/TSP patients, which was independent of the HAM/TSP disease signature. In conclusion, our results provide ex vivo mechanistic evidence for the observed immunovirological effect of in vivo IFN-ß treatment in HAM/TSP, reconcile an apparent IFN paradox in HTLV-1 research and identify biomarkers/targets for a precision medicine approach.

A genetic IFN/STAT1/FAS axis determines CD4 T stem cell memory levels and apoptosis in healthy controls and Adult T-cell Leukemia patients.

Adult T-cell leukemia (ATL) is an aggressive, chemotherapy-resistant CD4+CD25+ leukemia caused by HTLV-1 infection, which usually develops in a minority of patients several decades after infection. IFN + AZT combination therapy has shown clinical benefit in ATL, although its mechanism of action remains unclear. We have previously shown that an IFN-responsive FAS promoter polymorphism in a STAT1 binding site (rs1800682) is associated to ATL susceptibility and survival. Recently, CD4 T stem cell memory (TSCM) Fashi cells have been identified as the hierarchical cellular apex of ATL, but a possible link between FAS, apoptosis, proliferation and IFN response in ATL has not been studied. In this study, we found significant ex vivo antiproliferative, antiviral and immunomodulatory effects of IFN-α treatment in short-term culture of primary mononuclear cells from ATL patients (n = 25). Bayesian Network analysis allowed us to integrate ex vivo IFN-α response with clinical, genetic and immunological data from ATL patients, thereby revealing a central role for FAS -670 polymorphism and apoptosis in the coordinated mechanism of action of IFN-α. FAS genotype-dependence of IFN-induced apoptosis was experimentally validated in an independent cohort of healthy controls (n = 20). The same FAS -670 polymorphism also determined CD4 TSCM levels in a genome-wide twin study (p = 7 × 10-11, n = 460), confirming a genetic link between apoptosis and TSCM levels. Transcriptomic analysis and cell type deconvolution confirmed the FAS genotype/TSCM link and IFN-α-induced downregulation of CD4 TSCM-specific genes in ATL patient cells. In conclusion, ex vivo IFN-α treatment exerts a pleiotropic effect on primary ATL cells, with a genetic IFN/STAT1/Fas axis determining apoptosis vs. proliferation and underscoring the CD4 TSCM model of ATL leukemogenesis.

Target identification of small molecules using large-scale CRISPR-Cas mutagenesis scanning of essential genes.

Unraveling the mechanism of action and molecular target of small molecules remains a major challenge in drug discovery. While many cancer drugs target genetic vulnerabilities, loss-of-function screens fail to identify essential genes in drug mechanism of action. Here, we report CRISPRres, a CRISPR-Cas-based genetic screening approach to rapidly derive and identify drug resistance mutations in essential genes. It exploits the local genetic variation created by CRISPR-Cas-induced non-homologous end-joining (NHEJ) repair to generate a wide variety of functional in-frame mutations. Using large sgRNA tiling libraries and known drug-target pairs, we validate it as a target identification approach. We apply CRISPRres to the anticancer agent KPT-9274 and identify nicotinamide phosphoribosyltransferase (NAMPT) as its main target. These results present a powerful and simple genetic approach to create many protein variants that, in combination with positive selection, can be applied to reveal the cellular target of small-molecule inhibitors.

A Fashi Lymphoproliferative Phenotype Reveals Non-Apoptotic Fas Signaling in HTLV-1-Associated Neuroinflammation.

Human T-cell lymphotropic virus (HTLV)-1 was the first human retrovirus to be associated to cancer, namely adult T-cell leukemia (ATL), but its pathogenesis remains enigmatic, since only a minority of infected individuals develops either ATL or the neuroinflammatory disorder HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). A functional FAS -670 polymorphism in an interferon (IFN)-regulated STAT1-binding site has been associated to both ATL and HAM/TSP susceptibility. Fashi T stem cell memory (Tscm) cells have been identified as the hierarchical apex of ATL, but have not been investigated in HAM/TSP. In addition, both FAS and STAT1 have been identified in an IFN-inducible HAM/TSP gene signature, but its pathobiological significance remains unclear. We comprehensively explored Fas expression (protein/mRNA) and function in lymphocyte activation, apoptosis, proliferation, and transcriptome, in PBMC from a total of 47 HAM/TSP patients, 40 asymptomatic HTLV-1-infected individuals (AC), and 58 HTLV-1 -uninfected healthy controls. Fas surface expression followed a two-step increase from HC to AC and from AC to HAM/TSP. In HAM/TSP, Fas levels correlated positively to lymphocyte activation markers, but negatively to age of onset, linking Fashi cells to earlier, more aggressive disease. Surprisingly, increased lymphocyte Fas expression in HAM/TSP was linked to decreased apoptosis and increased lymphoproliferation upon in vitro culture, but not to proviral load. This Fashi phenotype is HAM/TSP-specific, since both ex vivo and in vitro Fas expression was increased as compared to multiple sclerosis (MS), another neuroinflammatory disorder. To elucidate the molecular mechanism underlying non-apoptotic Fas signaling in HAM/TSP, we combined transcriptome analysis with functional assays, i.e., blocking vs. triggering Fas receptor in vitro with antagonist and agonist-, anti-Fas mAb, respectively. Treatment with agonist anti-Fas mAb restored apoptosis, indicating biased, but not defective Fas signaling in HAM/TSP. In silico analysis revealed biased Fas signaling toward proliferation and inflammation, driven by RelA/NF-κB. Correlation of Fas transcript levels with proliferation (but not apoptosis) was confirmed in HAM/TSP ex vivo transcriptomes. In conclusion, we demonstrated a two-step increase in Fas expression, revealing a unique Fashi lymphocyte phenotype in HAM/TSP, distinguishable from MS. Non-apoptotic Fas signaling might fuel HAM/TSP pathogenesis, through increased lymphoproliferation, inflammation, and early age of onset.

Inflammatory Gene Expression Profile and Defective Interferon-γ and Granzyme K in Natural Killer Cells From Systemic Juvenile Idiopathic Arthritis Patients.

OBJECTIVE: Systemic juvenile idiopathic arthritis (JIA) is an immunoinflammatory disease characterized by arthritis and systemic manifestations. The role of natural killer (NK) cells in the pathogenesis of systemic JIA remains unclear. The purpose of this study was to perform a comprehensive analysis of NK cell phenotype and functionality in patients with systemic JIA. METHODS: Transcriptional alterations specific to NK cells were investigated by RNA sequencing of highly purified NK cells from 6 patients with active systemic JIA and 6 age-matched healthy controls. Cytokines (NK cell-stimulating and others) were quantified in plasma samples (n = 18). NK cell phenotype and cytotoxic activity against tumor cells were determined (n = 10), together with their interferon-γ (IFNγ)-producing function (n = 8). RESULTS: NK cells from the systemic JIA patients showed an altered gene expression profile compared to cells from the healthy controls, with enrichment of immunoinflammatory pathways, increased expression of innate genes including TLR4 and S100A9, and decreased expression of immune-regulating genes such as IL10RA and GZMK. In the patients' plasma, interleukin-18 (IL-18) levels were increased, and a decreased ratio of IFNγ to IL-18 was observed. NK cells from the patients exhibited specific alterations in the balance of inhibitory and activating receptors, with decreased killer cell lectin-like receptor G1 and increased NKp44 expression. Although NK cells from the patients showed increased granzyme B expression, consistent with intact cytotoxicity and degranulation against a tumor cell line, decreased granzyme K expression in CD56bright NK cells and defective IL-18-induced IFNγ production and signaling were demonstrated. CONCLUSION: NK cells are active players in the inflammatory environment typical of systemic JIA. Although their cytotoxic function is globally intact, subtle defects in NK-related pathways, such as granzyme K expression and IL-18-driven IFNγ production, may contribute to the immunoinflammatory dysregulation in this disease.