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

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

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

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

Immune cell profiling of the cerebrospinal fluid enables the characterization of the brain metastasis microenvironment

Brain metastases are the most common tumor of the brain with a dismal prognosis. A fraction of patients with brain metastasis benefit from treatment with immune checkpoint inhibitors (ICI) and the degree and phenotype of the immune cell infiltration has been used to predict response to ICI. However, the anatomical location of brain lesions limits access to tumor material to characterize the immune phenotype. Here, we characterize immune cells present in brain lesions and matched cerebrospinal fluid (CSF) using single-cell RNA sequencing combined with T cell receptor genotyping. Tumor immune infiltration and specifically CD8 + T cell infiltration can be discerned through the analysis of the CSF. Consistently, identical T cell receptor clonotypes are detected in brain lesions and CSF, confirming cell exchange between these compartments. The analysis of immune cells of the CSF can provide a non-invasive alternative to predict the response to ICI, as well as identify the T cell receptor clonotypes present in brain metastasis. The use of CSF for diagnosis of metastatic brain tumors could be of clinical and patient benefit. Here the authors undertake a single-cell RNA analysis of CSF and brain to determine whether the phenotype in the CSF is reflective of the phenotype in the tumo

LIF regulates CXCL9 in tumor-associated macrophages and prevents CD8+ T cell tumor-infiltration impairing anti-PD1 therapy

Càncer; Macròfags associats al tumor: LIF; CD8Cáncer; Macrófagos asociados al tumor; CD8Cancer; Tumor-associated macrophages; CD8Cancer response to immunotherapy depends on the infiltration of CD8+ T cells and the presence of tumor-associated macrophages within tumors. Still, little is known about the determinants of these factors. We show that LIF assumes a crucial role in the regulation of CD8+ T cell tumor infiltration, while promoting the presence of protumoral tumor-associated macrophages. We observe that the blockade of LIF in tumors expressing high levels of LIF decreases CD206, CD163 and CCL2 and induces CXCL9 expression in tumor-associated macrophages. The blockade of LIF releases the epigenetic silencing of CXCL9 triggering CD8+ T cell tumor infiltration. The combination of LIF neutralizing antibodies with the inhibition of the PD1 immune checkpoint promotes tumor regression, immunological memory and an increase in overall survival

Endothelial and myointimal cell functions related to vascular inflammation and remodeling in Giant-Cell Arteritis: Contribution of interleukin-23p19, interleukin-35 and endothelin-1

[eng] Giant cell arteritis (GCA) is vascular inflammatory disease frequently involving large and medium sized arteries. GCA pathogenesis is not completely characterized, although there is a well accepted general model based on the observation of the histopathology and supported by immunopathology studies. According to this model, CD4+T cells subsets (mainly Th1 and Th17) and macrophages are essential to orchestrate the amplification of pro-inflammatory cascades leading to chronic inflammation of the vessel wall. The main sites of entry of infiltrating leukocytes are the activated endothelial cells (EC) from the adventitial vasa vasorum. Importantly, the secretion of proteases by macrophages among others, promotes elastic lamina degradation and migration of leukocytes to the inner artery layers. Vascular smooth muscle cells (VSMC) have also an important role in GCA pathogenesis since, in this scenario; they acquire a pro-inflammatory phenotype by overexpressing adhesion molecules and pro-inflammatory cytokines, contributing to the persistence of inflammation. Inflammatory cell products, as well as vascular injury, trigger a vascular remodelling process which leads to the development of intimal hyperplasia and vascular lumen occlusion. VSMC acquisition of proliferative and migratory phenotype may contribute to vascular remodeling. Further investigation of molecules involved in GCA pathogenesis is needed in order to find new strategies to improve the current GCA treatment (glucocorticoids) since 50% of the patients relapse and 40% suffer form early (visual loss or stroke) or late (aortic aneurysm) complications derived from maladaptive vascular remodeling. For this reason, the aim of this doctoral thesis was to contribute knowledge to these GCA challenges by investigating new roles of two important molecules involved in the persistence of inflammation (IL-23p19 and IL-35) as well as a new role of endothelin 1 (ET-1) in GCA vascular remodeling. The IL-12 superfamily of cytokines is composed by four heterodimeric cytokines: IL-12 (p40 + p19), IL-23 (p40 ad p19), IL-27 (p28 and Ebi3) and IL-35 (Ebi3 and p35). Regarding to these cytokines, we have demonstrated a dichotomy in the expression of p19 and p40 subunits suggesting a role for p19 independent from p40 heterodimerization. We observed that p19 but not p40 was induced by a pro- inflammatory context in EC from the adventitial vasa vasorum of GCA specimens. Moreover, p19 was not secreted by stable p19-expressing EC suggesting an intracellular role. Our studies showed that p19 contributed to leukocyte attachment and transmigration by promoting overexpression of adhesion molecules ICAM-1 and VCAM-1. We also demonstrated that these effects were triggered by intracellular p19 binding to gp130 receptor, subsequently promoting phoshporylation and nuclear translocation of STAT3. IL-35 cytokine was also investigated as a consequence of the previous observation that p35 (but not p40) was constitutively expressed by VSMC of GCA specimens. This dichotomy suggested that p35 may have and independent role from that derived from p40 heterodimerization or that p35 may have another partner to form a cytokine different from IL-12. Our results demonstrated that p35 dimerizes with Ebi3 subunit in order to form IL-35 in GCA specimens. Interestingly, we found that IL-35 was mainly produced by leukocytes and VSMC in the adventitial/media junctions of GCA lesions. In order to mimic this context, we used co-cultures of temporal artery-derived primary VSMC together with leukocytes which demonstrated that IL-35 expression in VSMC was increased by pro-inflammatory cytokines IL-1β, TNFα and IFNg. In addition, we have demonstrated a new role for this cytokine in VSMC. IL-35 interacts with gp130 in order to promote pro-inflammatory phenotype of VSMC. This is an important observation since, until present; IL-35 has been described as an anti-inflammatory cytokine involved in regulatory T cell (Treg) differentiation. We previously reported that endothelin-1 (ET-1) and its receptors ETAR and ETBR are over-expressed in GCA lesions and GCA patients with ischemic complications have increased serum concentrations of ET-1. Although it has been observed that ET-1 may contribute to myofibroblast differentiation of fibroblasts, a crucial step in lung and skin fibrogenic diseases, the most investigated function of ET-1 in VSMC has been vascular tone regulation. In this thesis, we have demonstrated that ET-1 may contribute to vascular occlusion, by promoting the migration of VSMC through activation of the focal adhesion kinase (FAK)/Src and PI3K axis. In GCA lesions ET-1 was mainly produced by inflammatory cells and the interaction with PBMC increased ET-1 receptor expression by VSMC. ET-1 stimulated VSMC migration by inducing focal adhesion kinase (FAK) phosphorylation at Y397 permitting association with p85 subunit of PI3Kinase which co-localized with FAK at the cell protrusions of migrating cells. Accordingly, inhibition of both FAK and PI3K abrogated ET-1 induced migration. Consistently, blockade of ETAR and ETBR with BQ123 and BQ788 antagonists inhibited ET-1 induced VSMC migration and VSMC outgrowth from cultured GCA-arteries.[cat] L’arteritis de cèl·lules gegants (ACG) és una malaltia inflamatòria crònica d’etiologia desconeguda que majoritàriament afecta les arteries de mitjà i gran calibre, entre elles l’artèria temporal. El tractament actual de l’ACG són els glucocorticoides tot i que el 50 % dels pacients recauen. Els mecanismes implicats en l’ACG s’han anat caracteritzant a partir d’observacions histopatològiques de l’artèria temporal així com a partir d’estudis immunopatològics. S’observa un clar infiltrat inflamatori format majoritàriament per limfòcits CD4+T i macròfags que van migrant des de l’adventícia fins a la íntima. La porta d’entrada d’aquest infiltrat es creu que podrien ser els petits vasos adventicials (vasa vasorum) que en condicions normals nodreixen l’artèria i, en canvi, en condicions patològiques s’observa una clara activació de les cèl·lules endotelials que els composen. Les cèl·lules infiltrants secreten citoquines i quimiocines encarregades de reclutar constantment més leucòcits donant lloc a la persistència de la inflamació en el teixit arterial. A més, aquestes cèl·lules també interaccionen amb altres components de la paret arterial com les cèl·lules muscular llises (VSMC) que també adquireixen un fenotip pro-inflamatori. Per altra banda, en l’ACG també s’observa un important remodelat vascular patològic, majoritàriament degut al trencament de les làmines elàstiques de l’artèria i la migració cap a la llum vascular tant de l’infiltrat inflamatori com de les VSMC que adquireixen un fenotip més proliferatiu, migratori i productor de matriu extracel·lular que ajudarà a crear la hiperplàsia de la íntima, originant l’oclusió vascular. Tenint en compte aquest context, en aquesta tesi doctoral s’han estudiat dues molècules implicades en la persistència de la inflamació. Per una banda, l’estudi de la subunitat p19 de la IL-23. En condicions pro-inflamatòries aquesta molècula podria actuar independentment de la dimerització amb la p40 per formar la IL-23 i podria intervenir desde el compartiment intracel.lular a traves d’una interacció directa amb gp130, en l’activació de les cèl·lules endotelials que formen el vasa vasorum adventicial, ajudant així al reclutament leucocitari. Per altra banda, l’estudi de la IL-35, expressada per les VSMC i els leucòcits infiltrants i no només per les cèl·lules T reguladores com s’havia descrit anteriorment. Hem observat i descrit per primera vegada, que la IL-35 també estaria involucrada en la persistència de la inflamació de l’ACG, induint un fenotip pro-inflamatori en les VSMC. Pel que fa al remodelat vascular, hem estudiat l’endotelina (ET-1), que a través dels seus dos receptors (ETAR i ETBR) provocaria una inducció de la migració de les VSMC, tenint un rol essencial en la creació de la hiperplàsia de la íntima i la oclusió vascular

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

BackgroundGiant-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.ObjectivesThe 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 resultsp40 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 &lt; 0.0001 and 20.32 ± 21.78 vs 4.17 ± 4.43 relative units; p &lt; 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.ConclusionIL-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

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