Blocking GM-CSF receptor α with mavrilimumab reduces infiltrating cells, pro-inflammatory markers and neoangiogenesis in ex vivo cultured arteries from patients with giant cell arteritis

Effective and safe therapies are needed for the treatment of patients with giant cell arteritis (GCA). Emerging as a key cytokine in inflammation, granulocyte-macrophage colony stimulating factor (GM-CSF) may play a role in promoting inflammation in GCA.To investigate expression of GM-CSF and its receptor in arterial lesions from patients with GCA. To analyse activation of GM-CSF receptor-associated signalling pathways and expression of target genes. To evaluate the effects of blocking GM-CSF receptor α with mavrilimumab in ex vivo cultured arteries from patients with GCA.Quantitative real time PCR, in situ RNA hybridisation, immunohistochemistry, immunofluorescence and confocal microscopy, immunoassay, western blot and ex vivo temporal artery culture.GM-CSF and GM-CSF receptor α mRNA and protein were increased in GCA lesions; enhanced JAK2/STAT5A expression/phosphorylation as well as increased expression of target genes CD83 and Spi1/PU.1 were observed. Treatment of ex vivo cultured GCA arteries with mavrilimumab resulted in decreased transcripts of CD3ε, CD20, CD14 and CD16 cell markers, and reduction of infiltrating CD16 and CD3ε cells was observed by immunofluorescence. Mavrilimumab reduced expression of molecules relevant to T cell activation (human leukocyte antigen-DR [HLA-DR]) and Th1 differentiation (interferon-γ), the pro-inflammatory cytokines: interleukin 6 (IL-6), tumour necrosis factor α (TNFα) and IL-1β, as well as molecules related to vascular injury (matrix metalloprotease 9, lipid peroxidation products and inducible nitric oxide synthase [iNOS]). Mavrilimumab reduced CD34 + cells and neoangiogenesis in GCA lesions.The inhibitory effects of mavrilimumab on multiple steps in the GCA pathogenesis cascade in vitro are consistent with the clinical observation of reduced GCA flares in a phase 2 trial and support its development as a therapeutic option for patients with GCA.© Author(s) (or their employer(s)) 2022. Re-use permitted under CC BY. Published by BMJ

Endothelin-1 promotes vascular smooth muscle cell migration across the artery wall: a mechanism contributing to vascular remodelling and intimal hyperplasia in giant-cell arteritis

Background: Giant-cell arteritis (GCA) is an inflammatory disease of large/medium-sized arteries, frequently involving the temporal arteries (TA). Inflammation-induced vascular remodelling leads to vaso-occlusive events. Circulating endothelin-1 (ET1) is increased in patients with GCA with ischaemic complications suggesting a role for ET-1 in vascular occlusion beyond its vasoactive function. Objective: To investigate whether ET-1 induces a migratory myofibroblastic phenotype in human TAderived vascular smooth muscle cells (VSMC) leading to intimal hyperplasia and vascular occlusion in GCA. Methods and results: Immunofluorescence/confocal microscopy showed increased ET-1 expression in GCA lesions compared with control arteries. In inflamed arteries, ET-1 was predominantly expressed by infiltrating mononuclear cells whereas ET receptors, particularly ET-1 receptor B (ETB R), were expressed by both mononuclear cells and VSMC. ET-1 increased TA-derived VSMC migration in vitro and α-smooth muscle actin (αSMA) expression and migration from the media to the intima in cultured TA explants. ET-1 promoted VSMC motility by increasing activation of focal adhesion kinase (FAK), a crucial molecule in the turnover of focal adhesions during cell migration. FAK activation resulted in Y397 autophosphorylation creating binding sites for Src kinases and the p85 subunit of PI3kinases which, upon ET-1 exposure, colocalised with FAK at the focal adhesions of migrating VSMC. Accordingly, FAK or PI3K inhibition abrogated ET-1-induced migration in vitro. Consistently, ET-1 receptor A and ETB R antagonists reduced αSMA expression and delayed VSMC outgrowth from cultured GCA-involved artery explants. Conclusions: ET-1 is upregulated in GCA lesions and, by promoting VSMC migration towards the intimal layer, may contribute to intimal hyperplasia and vascular occlusion in GCA

Positron emission tomography assessment of large vessel inflammation in patients with newly diagnosed, biopsy-proven giant cell arteritis: a prospective, case-control study

BACKGROUND: Positron emission tomography (PET) scan is emerging as a promising imaging technique to detect large-vessel inflammation in giant cell arteritis (GCA). However, the lack of a standardised definition of arteritis based on (18)fluorodeoxyglucose (FDG) uptake is an important limitation to the use of PET scan for diagnostic purposes. OBJECTIVE: To prospectively assess the intensity and distribution of FDG uptake at different vascular territories in patients with newly diagnosed GCA compared with controls. METHODS: 32 consecutive, biopsy-proven, GCA patients treated with glucocorticoids for ≤3 days were included. The control group consisted of 20 individuals, who underwent PET/CT for cancer staging. Maximal standardised uptake value (SUVm) was calculated at four aortic segments, supraaortic branches and iliac-femoral territory. Sensitivity and specificity was calculated by receiver-operator characteristic curves (ROC) analysis. RESULTS: Mean SUVm was significantly higher in patients than in controls in all vessels explored and correlated with acute-phase reactants and serum IL-6. Mean of the SUVm at all the vascular territories had an area under the curve (AUC) of 0.830, and a cut-off of 1.89 yielded a sensitivity of 80% and a specificity of 79% for GCA diagnosis. There were no significant differences in AUC among the vascular beds examined. CONCLUSIONS: FDG uptake by large vessels has a substantial sensitivity and specificity for GCA diagnosis

Relapses in patients with giant-cell arteritis: prevalence, characteristics and associated clinical findings in a longitudinally followed cohort of 106 patients.

Giant cell arteritis (GCA) is a relapsing disease. However, the nature, chronology, therapeutic impact, and clinical consequences of relapses have been scarcely addressed. We conducted the present study to investigate the prevalence, timing, and characteristics of relapses in patients with GCA and to analyze whether a relapsing course is associated with disease-related complications, increased glucocorticoid (GC) doses, and GC-related adverse effects. The study cohort included 106 patients, longitudinally followed by the authors for 7.8 ± 3.3 years. Relapses were defined as reappearance of disease-related symptoms requiring treatment adjustment. Relapses were classified into 4 categories: polymyalgia rheumatica (PMR), cranial symptoms (including ischemic complications), systemic disease, or symptomatic large vessel involvement. Cumulated GC dose during the first year of treatment, time required to achieve a maintenance prednisone dose <10 mg/d (T10), <5 mg/d (T5), or complete prednisone discontinuation (T0), and GC-related side effects were recorded. Sixty-eight patients (64%) experienced at least 1 relapse, and 38 (36%) experienced 2 or more. First relapse consisted of PMR in 51%, cranial symptoms in 31%, and systemic complaints in 18%. Relapses appeared predominantly, but not exclusively, within the first 2 years of treatment, and only 1 patient developed visual loss. T10, T5, and T0 were significantly longer in patients with relapses than in patients without relapse (median, 40 vs 27 wk, p  < 0.0001; 163 vs 89.5 wk, p = 0.004; and 340 vs 190 wk, p = 0.001, respectively). Cumulated prednisone dose during the first year was significantly higher in relapsing patients (6.2 ± 1.7 g vs 5.4 ± 0.78 g, p = 0.015). Osteoporosis was more common in patients with relapses compared to those without (65% vs 32%, p = 0.001). In conclusion, the results of the present study provide evidence that a relapsing course is associated with higher and prolonged GC requirements and a higher frequency of osteoporosis in GCA

Blocking interferon γ reduces expression of chemokines CXCL9, CXCL10 and CXCL11 and decreases macrophage infiltration in ex vivo cultured arteries from patients with giant cell arteritis

BACKGROUND: Interferon γ (IFNγ) is considered a seminal cytokine in the pathogenesis of giant cell arteritis (GCA), but its functional role has not been investigated. We explored changes in infiltrating cells and biomarkers elicited by blocking IFNγ with a neutralising monoclonal antibody, A6, in temporal arteries from patients with GCA. METHODS: Temporal arteries from 34 patients with GCA (positive histology) and 21 controls were cultured on 3D matrix (Matrigel) and exposed to A6 or recombinant IFNγ. Changes in gene/protein expression were measured by qRT-PCR/western blot or immunoassay. Changes in infiltrating cells were assessed by immunohistochemistry/immunofluorescence. Chemotaxis/adhesion assays were performed with temporal artery-derived vascular smooth muscle cells (VSMCs) and peripheral blood mononuclear cells (PBMCs). RESULTS: Blocking endogenous IFNγ with A6 abrogated STAT-1 phosphorylation in cultured GCA arteries. Furthermore, selective reduction in CXCL9, CXCL10 and CXCL11 chemokine expression was observed along with reduction in infiltrating CD68 macrophages. Adding IFNγ elicited consistent opposite effects. IFNγ induced CXCL9, CXCL10, CXCL11, CCL2 and intracellular adhesion molecule-1 expression by cultured VSMC, resulting in increased PBMC chemotaxis/adhesion. Spontaneous expression of chemokines was higher in VSMC isolated from GCA-involved arteries than in those obtained from controls. Incubation of IFNγ-treated control arteries with PBMC resulted in adhesion/infiltration by CD68 macrophages, which did not occur in untreated arteries. CONCLUSIONS: Our ex vivo system suggests that IFNγ may play an important role in the recruitment of macrophages in GCA by inducing production of specific chemokines and adhesion molecules. Vascular wall components (ie, VSMC) are mediators of these functions and may facilitate progression of inflammatory infiltrates through the vessel wall

Expression and function of IL12/23 related cytokine subunits (p35, p40, and p19) in giant-cell arteritis lesions: contribution of p40 to Th1- and Th17-mediated inflammatory pathways

Background: Giant-cell arteritis (GCA) is considered a T helper (Th)1- and Th17-mediated disease. Interleukin (IL)-12 is a heterodimeric cytokine (p35/p40) involved in Th1 differentiation. When combining with p19 subunit, p40 compose IL-23, a powerful pro-inflammatory cytokine that maintains Th17 response. Objectives: The aims of this study were to investigate p40, p35, and p19 subunit expression in GCA lesions and their combinations to conform different cytokines, to assess the effect of glucocorticoid treatment on subunit expression, and to explore functional roles of p40 by culturing temporal artery sections with a neutralizing anti-human IL-12/IL-23p40 antibody. Methods and results: p40 and p19 mRNA concentrations measured by real-time RT-PCR were significantly higher in temporal arteries from 50 patients compared to 20 controls (4.35 ± 4.06 vs 0.51 ± 0.75; p < 0.0001 and 20.32 ± 21.78 vs 4.17 ± 4.43 relative units; p < 0.0001, respectively). No differences were found in constitutively expressed p35 mRNA. Contrarily, p40 and p19 mRNAs were decreased in temporal arteries from 16 treated GCA patients vs those from 34 treatment-naïve GCA patients. Accordingly, dexamethasone reduced p40 and p19 expression in cultured arteries. Subunit associations to conform IL-12 and IL-23 were confirmed by proximity-ligation assay in GCA lesions. Immunofluorescence revealed widespread p19 and p35 expression by inflammatory cells, independent from p40. Blocking IL-12/IL-23p40 tended to reduce IFNγ and IL-17 mRNA production by cultured GCA arteries and tended to increase Th17 inducers IL-1β and IL-6. Conclusion: IL-12 and IL-23 heterodimers are increased in GCA lesions and decrease with glucocorticoid treatment. p19 and p35 subunits are much more abundant than p40, indicating an independent role for these subunits or their potential association with alternative subunits. The modest effect of IL-12/IL-23p40 neutralization may indicate compensation by redundant cytokines or cytokines resulting from alternative combinations

Glucocorticoids—All-Rounders Tackling the Versatile Players of the Immune System

Glucocorticoids regulate fundamental processes of the human body and control cellular functions such as cell metabolism, growth, differentiation, and apoptosis. Moreover, endogenous glucocorticoids link the endocrine and immune system and ensure the correct function of inflammatory events during tissue repair, regeneration, and pathogen elimination via genomic and rapid non-genomic pathways. Due to their strong immunosuppressive, anti-inflammatory and anti-allergic effects on immune cells, tissues and organs, glucocorticoids significantly improve the quality of life of many patients suffering from diseases caused by a dysregulated immune system. Despite the multitude and seriousness of glucocorticoid-related adverse events including diabetes mellitus, osteoporosis and infections, these agents remain indispensable, representing the most powerful, and cost-effective drugs in the treatment of a wide range of rheumatic diseases. These include rheumatoid arthritis, vasculitis, and connective tissue diseases, as well as many other pathological conditions of the immune system. Depending on the therapeutically affected cell type, glucocorticoid actions strongly vary among different diseases. While immune responses always represent complex reactions involving different cells and cellular processes, specific immune cell populations with key responsibilities driving the pathological mechanisms can be identified for certain autoimmune diseases. In this review, we will focus on the mechanisms of action of glucocorticoids on various leukocyte populations, exemplarily portraying different autoimmune diseases as heterogeneous targets of glucocorticoid actions: (i) Abnormalities in the innate immune response play a crucial role in the initiation and perpetuation of giant cell arteritis (GCA). (ii) Specific types of CD4+ T helper (Th) lymphocytes, namely Th1 and Th17 cells, represent important players in the establishment and course of rheumatoid arthritis (RA), whereas (iii) B cells have emerged as central players in systemic lupus erythematosus (SLE). (iv) Allergic reactions are mainly triggered by several different cytokines released by activated Th2 lymphocytes. Using these examples, we aim to illustrate the versatile modulating effects of glucocorticoids on the immune system. In contrast, in the treatment of lymphoproliferative disorders the pro-apoptotic action of glucocorticoids prevails, but their mechanisms differ depending on the type of cancer. Therefore, we will also give a brief insight into the current knowledge of the mode of glucocorticoid action in oncological treatment focusing on leukemia

Analysis of the common genetic component of large-vessel vasculitides through a meta- Immunochip strategy

Giant cell arteritis (GCA) and Takayasu's arteritis (TAK) are major forms of large-vessel vasculitis (LVV) that share clinical features. To evaluate their genetic similarities, we analysed Immunochip genotyping data from 1,434 LVV patients and 3,814 unaffected controls. Genetic pleiotropy was also estimated. The HLA region harboured the main disease-specific associations. GCA was mostly associated with class II genes (HLA-DRB1/HLA-DQA1) whereas TAK was mostly associated with class I genes (HLA-B/MICA). Both the statistical significance and effect size of the HLA signals were considerably reduced in the cross-disease meta-analysis in comparison with the analysis of GCA and TAK separately. Consequently, no significant genetic correlation between these two diseases was observed when HLA variants were tested. Outside the HLA region, only one polymorphism located nearby the IL12B gene surpassed the study-wide significance threshold in the meta-analysis of the discovery datasets (rs755374, P?=?7.54E-07; ORGCA?=?1.19, ORTAK?=?1.50). This marker was confirmed as novel GCA risk factor using four additional cohorts (PGCA?=?5.52E-04, ORGCA?=?1.16). Taken together, our results provide evidence of strong genetic differences between GCA and TAK in the HLA. Outside this region, common susceptibility factors were suggested, especially within the IL12B locus

A genome-wide association study identifies risk alleles in plasminogen and P4HA2 associated with giant cell arteritis

Giant cell arteritis (GCA) is the most common form of vasculitis in individuals older than 50 years in Western countries. To shed light onto the genetic background influencing susceptibility for GCA, we performed a genome-wide association screening in a well-powered study cohort. After imputation, 1,844,133 genetic variants were analysed in 2,134 cases and 9,125 unaffected controls from ten independent populations of European ancestry. Our data confirmed HLA class II as the strongest associated region (independent signals: rs9268905, P = 1.94E-54, per-allele OR = 1.79; and rs9275592, P = 1.14E-40, OR = 2.08). Additionally, PLG and P4HA2 were identified as GCA risk genes at the genome-wide level of significance (rs4252134, P = 1.23E-10, OR = 1.28; and rs128738, P = 4.60E-09, OR = 1.32, respectively). Interestingly, we observed that the association peaks overlapped with different regulatory elements related to cell types and tissues involved in the pathophysiology of GCA. PLG and P4HA2 are involved in vascular remodelling and angiogenesis, suggesting a high relevance of these processes for the pathogenic mechanisms underlying this type of vasculitis

A Large-Scale Genetic Analysis Reveals a Strong Contribution of the HLA Class II Region to Giant Cell Arteritis Susceptibility

We conducted a large-scale genetic analysis on giant cell arteritis (GCA), a polygenic immune-mediated vasculitis. A case-control cohort, comprising 1,651 case subjects with GCA and 15,306 unrelated control subjects from six different countries of European ancestry, was genotyped by the Immunochip array. We also imputed HLA data with a previously validated imputation method to perform a more comprehensive analysis of this genomic region. The strongest association signals were observed in the HLA region, with rs477515 representing the highest peak (p = 4.05 × 10−40, OR = 1.73). A multivariate model including class II amino acids of HLA-DRβ1 and HLA-DQα1 and one class I amino acid of HLA-B explained most of the HLA association with GCA, consistent with previously reported associations of classical HLA alleles like HLA-DRB1∗04. An omnibus test on polymorphic amino acid positions highlighted DRβ1 13 (p = 4.08 × 10−43) and HLA-DQα1 47 (p = 4.02 × 10−46), 56, and 76 (both p = 1.84 × 10−45) as relevant positions for disease susceptibility. Outside the HLA region, the most significant loci included PTPN22 (rs2476601, p = 1.73 × 10−6, OR = 1.38), LRRC32 (rs10160518, p = 4.39 × 10−6, OR = 1.20), and REL (rs115674477, p = 1.10 × 10−5, OR = 1.63). Our study provides evidence of a strong contribution of HLA class I and II molecules to susceptibility to GCA. In the non-HLA region, we confirmed a key role for the functional PTPN22 rs2476601 variant and proposed other putative risk loci for GCA involved in Th1, Th17, and Treg cell function