INTRODUCTION:Anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) causes severe multisystemic organ damage. The main phenotypes, microscopic polyangiitis (MPA) and granulomatosis with polyangiitis (GPA), share similarities but differ in clinical presentation and outcome. To uncover their molecular differences, we performed transcriptomic profiling of kidney tissue, then focused on type I interferon (IFN-I) pathway activation in kidney and blood and its clinical implications. METHODS:We analyzed two independent cohorts (Maine-Anjou and RENVAS registries) totaling 193 patients with AAV and glomerulonephritis. NanoString nCounter transcriptomic profiling, and serum inflammatory molecules quantification were conducted. Comparative analyses of MPA vs. GPA (and MPO-AAV vs. PR3-AAV) were validated using independent public datasets (including kidney spatial transcriptomic datasets, European cDNA Renal Biobank and blood from the RAVE trial). RESULTS:The kidney IFN-I signature found in AAV-GN is upregulated in MPA/MPO-AAV compared to GPA/PR3-AAV and controls. Quantitative PCR, MxA immunohistochemistry, and analysis of external datasets confirmed these findings. This IFN-I signature, close to the one found in lupus nephritis, is linked to the extent of pDC infiltration. Kidney IFN-I activation correlated with increased kidney fibrosis, independently of kidney function. High kidney IFN-I signatures were linked to lower kidney survival, independently of kidney function and pathological scores. MPA kidneys also exhibited higher mast cell and T-cell infiltration. Systemic analyses showed elevated IFNα and interferon related inflammatory molecules in all patients with AAV, but a stronger IFN-I gene signature was found in immune cells from MPA. CONCLUSIONS:Our study identifies an IFN-I signature in AAV, especially in MPA/MPO-AAV, underscoring its potential role in disease heterogeneity and kidney pathology. IFN-I emerges as a potential prognostic biomarker and therapeutic target in AAV, particularly for MPA. Further studies are needed to clarify its mechanisms and explore IFN-I modulation in clinical trials.

Key PointsLarge-scale kidney transcriptomics identifies a 12-gene signature, including and , predicting kidney failure in ANCA-associated vasculitis.CLUC3This molecular signature outperformed Berden, renal risk score, and ANCA kidney risk score clinicopathologic classifications.ANCA-associated vasculitis with GN kidneys show broad immune dysregulation, notably in complement, TGF beta, and immunometabolism pathways.BackgroundANCA-associated vasculitis with GN (AAV-GN) frequently progresses to kidney failure. However, tools for risk stratification of kidney outcomes remain limited. Existing approaches inadequately capture the molecular complexity underlying kidney injury, despite its potential value to tailor therapeutic management. We explored whether kidney transcriptomics could identify molecular signatures linked to kidney outcomes. MethodsWe included 199 patients with AAV-GN from two multicenter biobanks, and 23 controls. Kidney biopsies were profiled using NanoString nCounter to assess the expression of 750 immune-related genes. We conducted differential gene expression analysis, pathway enrichment analysis, and immune cell infiltration estimation to explore associations with kidney function and survival. A 12-gene prognostic signature was developed through least absolute shrinkage and selection operator-penalized Cox regression and compared with established histologic classifications (Berden classification, renal risk score, and ANCA kidney risk score) with robust internal validation. ResultsAAV-GN demonstrated extensive immune dysregulation with 150 differentially expressed genes versus controls, highlighting complement activation, immune cell recruitment and activation, TGF beta signaling, and immunometabolism pathways. Immune cell infiltration was marked by increased macrophages, dendritic cells, neutrophils, and T-cell subsets, reflecting broad immune activation. Initial eGFR correlated with the expression of 319 genes. A 12-gene signature (CLU, C3, LTF, FLT1, PLCG2, FES, PRKCD, TXNIP, SLC7A5, PTEN, NRBF2, and NFATC1) was significantly more strongly associated with kidney survival than were established histologic classifications (adjusted P value < 0.0001). Both high expression and low expression of several immune pathways (especially lymphocyte trafficking) were associated with better outcomes compared with intermediate expression. ConclusionsTranscriptomic analysis of kidney biopsies in AAV-GN identified 150 differentially expressed immune-related genes and led to the development of a 12-gene signature that correlated strongly with kidney survival, outperforming established histologic classifications.

Background Several scores have been developed to predict mortality at anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) diagnosis. Their prognostic value in Caucasian patients with kidney involvement (AAV-GN) remains uncertain as none has been developed in this specific population. We aimed to propose a novel and more accurate score specific for them.Methods This multicentric study included patients diagnosed with AAV-GN since January 2000 in four nephrology centers (recorded in the Maine-Anjou AAV-GN Registry). Existing scores and baseline characteristics were assessed at diagnosis before any therapeutic intervention. A multivariable analysis was performed to build a new predictive score for death. Its prognosis performance (area under receiving operating curve and C-index) and accuracy (Brier score) was compared with existing scores. One hundred and eighty-five patients with AAV-GN from the RENVAS registry were used as a validation cohort.Results A total of 228 patients with AAV-GN from the Maine-Anjou registry were included to build the new score. It included the four components most associated with death: age, history of hypertension or cardiac disease, creatinine and hemoglobin levels at diagnosis. Overall, 194 patients had all the data available to determine the performance of the new score and existing scores. The new score performed better than the previous ones in the development and in the validation cohort. Among the scores tested, only Five-Factor Score and Japanese Vasculitis Activity Score had good performance in predicting death in AAV-GN.Conclusions This original score, named DANGER (Death in ANCA Glomerulonephritis-Estimating the Risk), may be useful to predict the risk of death in AAV-GN patients. Validation in different populations is needed to clarify its role in assisting clinical decisions. Graphical Abstract

Abstract Background and Aims Kidney involvement in ANCA-associated vasculitis (AAV-GN) predicts poor patient and kidney survival. Deciphering the transcriptomic landscape in AAV-GN may provide insights into pathogenic mechanisms or identify biomarkers for refining diagnosis and/or prognosis, which would help stratify risk and tailor therapeutic management. We aimed to investigate the potential prognostic value of kidney transcripts associated with kidney survival. Method This study included adult patients with AAV-GN from the French Maine-Anjou Registry. Immune gene transcript analysis was performed on RNA extracted from 97 AAV-GN kidney biopsies using NanoString technology. Transcripts of interest were selected, and their prognostic performance was assessed with respect to current histological-based classifications. Following the identification of a possible role for clusterin (CLU), the relationship between serum CLU and prognosis was assessed. Results Among the 750 evaluated transcripts, we identified a 4-gene signature (XRCC6, PRKCD, TEK, and CLU) that was strongly associated with kidney survival (Fig. 1A). This signature predicted kidney survival better than histological-based classifications (global C-Index 0.87 vs. 0.65 for Berden classification or 0.81 for Renal Risk Score, with better time-dependent AUC and Brier scores, especially beyond 1 year after diagnosis) (Fig. 1B). Among these 4 transcripts, the expression level of the CLU transcript had the highest correlation with glomerular involvement, kidney function at diagnosis, and kidney survival. Serum CLU levels were associated with kidney survival, especially when assessed at 6 months from diagnosis (Fig. 1C, P = .023). Conclusion Transcriptomic analysis of kidney biopsies of AAV-GN identified potential transcripts that may improve prediction of kidney survival. This transcriptomic signature may help us gain a deeper understanding of the AAV-GN pathogenesis and provide insights for developing new therapeutic options.

AbstractMacrophages fight infection and ensure tissue repair, often operating at nutrient-poor wound sites. We investigated the ability of human macrophages to metabolize glycogen. We observed that the cytokines GM-CSF and M-CSF plus IL-4 induced glycogenesis and the accumulation of glycogen by monocyte-derived macrophages. Glyconeogenesis occurs in cells cultured in the presence of the inflammatory cytokines GM-CSF and IFNγ (M1 cells), via phosphoenolpyruvate carboxykinase 2 (PCK2) and fructose-1,6-bisphosphatase 1 (FBP1). Enzyme inhibition with drugs or gene silencing techniques and 13C-tracing demonstrate that glutamine (metabolized by the TCA cycle), lactic acid, and glycerol were substrates of glyconeogenesis only in M1 cells. Tumor-associated macrophages (TAMs) also store glycogen and can perform glyconeogenesis. Finally, macrophage glycogenolysis and the pentose phosphate pathway (PPP) support cytokine secretion and phagocytosis regardless of the availability of extracellular glucose. Thus, glycogen metabolism supports the functions of human M1 and M2 cells, with inflammatory M1 cells displaying a possible dependence on glyconeogenesis.

Background: The absorption of vitamin B12 is hindered in pernicious anemia (PA) owing to intrinsic factor deficiency. Traditionally, intramuscular vitamin B12 injections were the standard treatment, bypassing the impaired absorption. Although there is potential for oral vitamin B12 supplementation through passive enteral absorption, it is not commonly prescribed in PA owing to limited studies assessing its efficacy. Objectives: We aimed to assess the efficacy of oral vitamin B12 supplementation in PA. Methods: We enrolled participants diagnosed with incident vitamin B12 deficiency related to PA. The diagnosis of PA was based on the presence classical immune gastritis and of anti -intrinsic factor and/or antiparietal cell antibodies. To evaluate the vitamin B12 status, we measured total plasma vitamin B12, plasma homocysteine, and plasma methylmalonic acid (pMMA) concentration and urinary methylmalonic acid - to - creatinine ratio. Participants were treated with oral cyanocobalamin at a dosage of 1000 mu g/d throughout the study duration. Clinical and biological vitamin B12 de fi ciency related features were prospectively and systematically assessed over the 1-y study duration. Results: We included 26 patients with vitamin B12 deficiency revealing PA. Following 1 mo of oral vitamin B12 supplementation, 88.5% of patients were no longer deficient in vitamin B12, with significant improvement of plasma vitamin B12 [407 (297 - 485) compared with 148 (116 - 213) pmol/L; P 0.0001], plasma homocysteine [13.5 (10.9 - 29.8) compared with 18.6 (13.7 - 46.8) mu mol/L; P < 0.0001], and pMMA [0.24 (0.16 - 0.38) compared with 0.56 (0.28 - 1.09) pmol/L; P < 0.0001] concentrations than those at baseline. The enhancement of these biological parameters persisted throughout the 12 month follow-up, with no patients showing vitamin B12 deficiency by the end of the follow-up period. The median time to reverse initial vitamin B12 deficiency abnormalities ranged from 1 mo for hemolysis to 4 mo for mucosal symptoms. Conclusions: Oral supplementation with 1000 mu g/d of cyanocobalamin has been shown to improve vitamin B12 deficiency in PA.

Mycosis fungoides (MF) is the most prevalent primary cutaneous T -cell lymphoma, with an indolent or aggressive course and poor survival. The pathogenesis of MF remains unclear, and prognostic factors in the early stages are not well established. Here, we characterized the most recurrent genomic alterations using whole-exome sequencing of 67 samples from 48 patients from Lille University Hospital (France), including 18 sequential samples drawn across stages of the malignancy. Genomic data were analyzed on the Broad Institute 's Terra bioinformatics platform. We found that gain7q, gain10p15.1 ( IL2RA and IL15RA ), del10p11.22 ( ZEB1 ), or mutations in JUNB and TET2 are associated with high -risk disease stages. Furthermore, gain7q, gain10p15.1 ( IL2RA and IL15RA ), del10p11.22 ( ZEB1 ), and del6q16.3 ( TNFAIP3 ) are coupled with shorter survival. Del6q16.3 ( TNFAIP3 ) was a risk factor for progression in patients at low risk. By analyzing the clonal heterogeneity and the clonal evolution of the cohort, we de fined different phylogenetic pathways of the disease with acquisition of JUNB , gain10p15.1 ( IL2RA and IL15RA ), or del12p13.1 ( CDKN1B ) at progression. These results establish the genomics and clonality of MF and identify potential patients at risk of progression, independent of their clinical stage.

Background: Bidirectional interactions between eosinophils and mast cells (MCs) have been reported in various allergic diseases. Bone marrow (BM) eosinophilia, and to a lesser extent blood eosinophilia, is common in systemic mastocytosis (SM), but its significance remains unknown. Objective: We described blood and BM eosinophil characteristics in SM. Methods: A large collection of BM biopsy samples was analyzed using immunohistochemical staining and whole-slide imaging. Eosinophil and extracellular granules were detected by eosinophil peroxidase (EPX) staining and MCs by KIT staining. Complementary analyses were conducted using flow cytometry and immunofluorescence. Results: Eosinophil infiltrates and large areas of eosinophil degranulation were observed within or around BM MC infiltrates in SM. EPX staining surface, highlighting intact eosinophils and eosinophil degranulation, was higher in nonadvanced SM (n = 37 BM biopsy samples) compared with both controls (n = 8, P = .0003) and advanced SM (n = 24, P = .014). In nonadvanced SM, positive correlations were observed between serum tryptase levels and percentages of eosinophil counts in BM aspirations (Spearman r coefficient r = 0.38, P = .038), eosinophils count in BM biopsy samples (r = 0.45, P = .007), EPX staining (r = 0.37, P = .035), and eosinophil degranulation (r = 0.39, P = .023). Eosinophil counts in BM biopsy samples also correlated with MC counts (r = 0.47, P = .006) and KIT staining surface (r = 0.49, P = .003). BM MCs expressed IL-5 receptor and other usual eosinophil cytokine/chemokine receptors, and blood eosinophils displayed several increased surface markers compared with controls, suggesting an activated state. Conclusion: Our data suggest possible cross talk between MCs and eosinophils, supporting MC tryptase release and MC activation-related symptoms. This suggests a rationale for targeting eosinophils in nonadvanced SM not fully controlled by other therapies. (J Allergy Clin Immunol 2024;154:1523-33.)

Supplementary Figure S3: Expression vector, mutagenesis oligonucleotides, and antibodies used for the functional analysis of the 8 somatic mutations in RTKs

ABSTRACT Background Kidney injury molecule 1 (KIM-1) is a transmembrane glycoprotein expressed by proximal tubular cells, recognized as an early, sensitive and specific urinary biomarker for kidney injury. Blood KIM-1 was recently associated with the severity of acute and chronic kidney damage but its value in antineutrophil cytoplasmic antibodies (ANCA)-associated vasculitis with glomerulonephritis (ANCA-GN) has not been studied. Thus, we analyzed its expression at ANCA-GN diagnosis and its relationship with clinical presentation, kidney histopathology and early outcomes. Methods We assessed KIM-1 levels and other pro-inflammatory molecules (C-reactive protein, interleukin-6, tumor necrosis factor α, monocyte chemoattractant protein-1 and pentraxin 3) at ANCA-GN diagnosis and after 6 months in patients included in the Maine-Anjou registry, which gathers data patients from four French Nephrology Centers diagnosed since January 2000. Results Blood KIM-1 levels were assessed in 54 patients. Levels were elevated at diagnosis and decreased after induction remission therapy. KIM-1 was associated with the severity of renal injury at diagnosis and the need for kidney replacement therapy. In opposition to other pro-inflammatory molecules, KIM-1 correlated with the amount of acute tubular necrosis and interstitial fibrosis/tubular atrophy (IF/TA) on kidney biopsy, but not with interstitial infiltrate or with glomerular involvement. In multivariable analysis, elevated KIM-1 predicted initial estimated glomerular filtration rate (β = –19, 95% CI –31, –7.6, P = .002). Conclusion KIM-1 appears as a potential biomarker for acute kidney injury and for tubulointerstitial injury in ANCA-GN. Whether KIM-1 is only a surrogate marker or is a key immune player in ANCA-GN pathogenesis remain to be determined.

Supplementary Figure S7: Fibroblast transformation with each of the 8 somatic-mutation-harboring RTKs

Abstract Background and Aims Antineutrophil-cytoplasmic antibodies (ANCA)-associated vasculitis (AAV) with kidney involvement (AAV-GN) frequently evolves to end-stage kidney disease (ESKD) despite aggressive immunosuppressive treatment. Several risk scores have been used to assess renal prognosis. We aimed to determine whether kidney function and markers of AAV-GN activity after 6 months could improve the prediction of ESKD. Method This retrospective and observational study included adult patients with AAV-GN recruited from 6 French nephrology centers (including from the Maine-Anjou AAV registry). The primary outcome was kidney survival. Analyses were conducted in the whole population and in a sub-population that did not develop ESKD early in the course of the disease. Results 241 patients were included in the whole cohort, with a median follow-up of 59 months. At diagnosis, Berden classification and Renal Risk Score (RRS) were not found better than kidney function (eGFR) alone, at predicting ESKD (C-index = 0.70, 0.79, 0.82, respectively). At 6 months, 20 patients reached ESKD. In the sub-population of 221 patients, 6 months eGFR outperformed Berden classification and RRS (C-index = 0.88, 0.62, 0.69, respectively) to predict ESKD. RRS performed better when it was updated with the eGFR at 6 months instead of the baseline eGFR. While 6-months proteinuria was associated with ESKD and improved ESKD prediction, hematuria and serological remission did not. Conclusion This work suggests the interest of the reassessment of the renal prognosis 6 months after AAV-GN diagnosis. Kidney function at this time remains the most reliable for predicting renal outcome. To improve the long-term prediction of ESKD, there may be a place for repeated kidney biopsy following induction treatment, in order to obtain a precise evaluation of disease activity, discuss the immunosuppressive treatment strategy and improve prognosis scores.

Supplementary Figure S6: Amino acid sequence alignment of the VEGFR1 and VEGFR3 kinase domains

ABSTRACT Background Anti-neutrophil cytoplasmic antibody (ANCA) kinetic in ANCA-associated vasculitis with glomerulonephritis (AAV-GN) has been suggested to be associated with AAV relapse. Few studies have focused on its association with renal prognosis. Thus we aimed to investigate the relationship between ANCA specificity and the evolutive profile and renal outcomes. Methods This multicentric retrospective study included patients diagnosed with ANCA-GN since 1 January 2000. Patients without ANCA at diagnosis and with fewer than three ANCA determinations during follow-up were excluded. We analysed estimated glomerular filtration rate (eGFR) variation, renal-free survival and relapse-free survival according to three ANCA profiles (negative, recurrent and persistent) and to ANCA specificity [myeloperoxidase (MPO) or proteinase 3 (PR3)]. Results Over a follow-up of 56 months [interquartile range (IQR) 34–101], a median of 19 (IQR 13–25) ANCA determinations were performed for the 134 included patients. Patients with a recurrent/persistent ANCA profile had a lower relapse-free survival (P = .019) and tended to have a lower renal survival (P = .053) compared with those with a negative ANCA profile. Patients with a recurrent/persistent MPO-ANCA profile had the shortest renal survival (P = .015) and those with a recurrent/persistent PR3-ANCA profile had the worst relapse-free survival (P = .013) compared with other profiles. The negative ANCA profile was associated with a greater eGFR recovery. In multivariate regression analysis, it was an independent predictor of a 2-fold increase in eGFR at 2 years [odds ratio 6.79 (95% confidence interval 1.78–31.4), P = .008]). Conclusion ANCA kinetic after an ANCA-GN diagnosis is associated with outcomes. MPO-ANCA recurrence/persistence identifies patients with a lower potential of renal recovery and a higher risk of kidney failure, while PR3-ANCA recurrence/persistence identifies patients with a greater relapse risk. Thus ANCA kinetics may help identify patients with a smouldering disease.

Supplementary Figure S2: Validation of 9 somatic mutations in RTKs by Sanger sequencing

MET exon 14 skipping mutation is a hepatocyte growth factor (HGF)-dependent oncogenic driver in vitro and in humanized HGF knock-in mice.

Les interactions entre le polynucléaire éosinophile et le mastocyte ont été décrites dans des maladies inflammatoires et/ou allergiques variées (Galdiero et al., 2017). L’éosinophilie médullaire est une caractéristique classique de la biopsie ostéomédullaire (BOM) de la mastocytose systémique, mais le rôle potentiel des éosinophiles n’a jamais été exploré. Nous avons analysé 61 BOM de patients avec mastocytoses indolentes (n = 33), « smouldering » (n = 4), ou avancées (n = 24), ou contrôles (n = 8). Des immunomarquages anti-EPX (éosinophiles), anti-c-KIT (mastocytes) ou des doubles immunomarquages EPX/c-KIT ont été réalisés en immunohistochimie (IHC) et en immunofluorescence (IF) avec microscopie confocale. Les surfaces d’immunomarquages c-KIT ou EPX sont exprimées en pourcentages par rapport aux surfaces cellulaires non marquées : par exemple, la surface d’immunomarquage EPX est calculée comme la surface des infiltrats cellulaires marqués EPX/(somme des surfaces EPX+ et des surfaces non marquées EPX). L’expression de marqueurs de surface par les mastocytes médullaires a été analysée en cytométrie en flux (CMF), et des images en IF ont été utilisées pour conforter l’analyse de l’expression du récepteur de l’IL-5. L’expression de marqueurs de surface par les éosinophiles sanguins a été analysée en CMF, et des analyses moléculaires par NGS ont été réalisées sur éosinophiles triés. En utilisant une analyse automatisée non supervisée des immunomarquages sur lames entières, nous avons observé une surface d’immunomarquage EPX, représentant à la fois les infiltrats d’éosinophiles intacts et les éosinophiles dégranulés, plus importante dans les mastocytoses non avancées (ISM + SSM) (médiane [IQR] : 37 % [19 ; 61]) que dans les contrôles (7 % [2,25 ; 17,25], p = 0,0003) et mastocytoses avancées (17 % [9,25 ; 36,5], p = 0,0014). Dans les mastocytoses non avancées, nous avons observé des corrélations positives entre les taux de tryptase sérique et le pourcentage d’éosinophiles au myélogramme (coefficient r de Spearman de 0,38, p = 0,038), le pourcentage d’éosinophiles sur les BOM (r 0,45, p = 0 0,007), la surface d’immunomarquage EPX (r 0,37, p = 0,035) et le niveau de dégranulation des éosinophiles (r 0,39, p = 0,023). Le nombre d’éosinophiles sur les BOM était également corrélé avec le nombre de mastocytes (r 0,47, p = 0,006) et la surface d’immunomarquage c-KIT sur les BOM (r 0,49, p = 0,003). Aucun de ces résultats n’était observé dans les mastocytoses avancées. Dans les mastocytoses non avancées, nous avons observé en IHC et en IF que les infiltrats d’éosinophiles et de larges plages de dégranulation étaient présents au sein et autour des infiltrats mastocytaires. En CMF, les mastocytes médullaires exprimaient à leur surface les récepteurs des interleukines (IL) 5 (confirmé en IF), IL-9 et IL-3, et les récepteurs de chimiokines PGD2, éotaxines et TARC, qui pourraient contribuer aux interactions réciproques avec les éosinophiles. De plus, les données de NGS suggèrent que les hyperéosinophilies sanguines ne soient pas d’origine clonale dans la mastocytoses non avancées : chez 4 patients avec mastocytose indolente et hyperéosinophilie > 1500/mm3, 1 patiente n’avait pas de mutation c-KIT, 3 patients étaient mutés D816 V, et la fréquence allélique était dans ce cas < 5 % dans les éosinophiles triés, sans autre mutation sur-représentée dans les éosinophiles par rapport à l’ADN médullaire total. Enfin, les éosinophiles sanguins analysés en CMF ont un profil d’activation caractérisé par une augmentation des intégrines CD44 et CD62L, et de CD137/4-1BB. Les nombreux médiateurs qui peuvent activer et/ou attirer les 2 cellules pourraient expliquer ces colocalisations et les corrélations observées entre des paramètres liés au mastocyte (tryptase sérique, nombre de mastocytes...) et à l’éosinophile (nombre de cellules, niveau de dégranulation...). Une efficacité potentielle des anti-IL-5 dans la mastocytose systémique indolente et le syndrome d’activation mastocytaire est d’ailleurs suggérée (De Wilde et al., 2016 ;Guillet et al., 2021). Nos données, confrontées aux propriétés connues des éosinophiles et des mastocytes, suggèrent que les deux populations cellulaires pourraient interagir dans les mastocytoses non avancées. Ainsi, les éosinophiles pourraient contribuer à la production de tryptase par les mastocytes et contribuer aux manifestations cliniques liées à l’activation mastocytaire. Des essais cliniques évaluant le bénéfice et la tolérance des anticorps monoclonaux anti-IL-5 ou anti-IL-5R semblent à envisager dans les mastocytoses non avancées.