42 research outputs found
Regulació de l’activitat inflamatòria per IFN-gamma en l'Arteritis de Cèl·lules Gegants (ACG)
No full textL'ACG és una malaltia inflamatòria crònica de caràcter granulomatós, que afecta a les arteries grans i mitjanes en pacients d’edat avançada. El tractament majoritàriament establert per l'ACG són els glucocorticoides, que malgrat produir generalment una remissió ràpida dels símptomes, no eviten rebrots, i presenten una sèrie important d’efectes secundaris que fan necessària la investigació de teràpies alternatives. La investigació de nous tractaments requereix un coneixement més acurat sobre la patogènesi de la malaltia.
La fisiopatologia de la malaltia es fonamenta en hipòtesis i deduccions fetes a partir de l’observació de l’expressió de molècules que tenen funcions conegudes en altres patologies. Així doncs s’ha acceptat que l'ACG s'inicia amb la presentació d'un antigen desconegut per part de les cèl·lules dendrítiques. Això provocarà una onada inflamatòria amb activació de leucòcits, i la progressió d'un infiltrat inflamatori a través de la paret arterial desestructurant les diferents làmines, que conclou amb la hiperplàsia de la làmina íntima i la obturació de la llum del vas.
L'IFN-g per la seva banda, és una citoquina implicada en multitud de fenòmens relacionats amb la inflamació, com són l'activació de macròfags, la producció de molècules d’adhesió per part de les cèl·lules endotelials, o el reforçament de la via de diferenciació limfocitària Th1. Tots aquests fenòmens semblen rellevants durant la fisiopatologia de l'ACG, però malgrat això el rol de l'IFN-g en aquesta malaltia és encara desconegut.
Una de les limitacions que té la investigació en ACG, és l'absència d'un model animal que permeti la realització d'estudis funcionals. En aquest sentit, els objectius del nostre treball són la posada a punt d'un model de cultiu d'artèria que permeti avaluar els efectes de molècules bloquejants i/o fàrmacs amb finalitat terapèutica. I en segon lloc aprofundir en el paper de l'IFN-g en l'ACG, definint les funcions que pugui tenir en la fisiopatologia de la malaltia però també en el manteniment de d’infiltrat inflamatori a la paret arterial.
Així doncs, hem posat a punt el model de cultiu d'artèria temporal de pacients amb ACG, comprovant que l’estructura de la paret arterial es preserva durant temps perllongats en cultiu i mantenint la viabilitat de l'infiltrat inflamatori. Aquest model ens ha permès valorar les
diferències en l'expressió de molècules pro-inflamatòries i molècules involucrades en el remodelatge vascular, entre biòpsies positives i negatives, i quins efectes té el tractament amb glucocorticoides sobre l'expressió d’aquestes. Així doncs, les biòpsies de pacients amb ACG presenten nivells d'expressió més elevats de IFN-g, IL-1b, CCL3, 4 i 5, i MMP9. El tractament amb glucocorticoides disminueix l'expressió d’aquestes mateixes molècules i de IL-6, TNF-a i CXCL8, així com la presencia de macròfags (identificats mitjançant immunotinció amb CD68) a la paret arterial. En canvi els corticoides no afecten substancialment l’expressió de molècules involucrades en el remodelatge vascular com el PDGF, TGFb o col·làgens.
En el segon treball hem bloquejat l'IFN-g amb un anticòs monoclonal humà, que inhibeix en les artèries l’expressió i fosforilació de Stat-1, el factor de transcripció de la via canònica de l'IFN-g. Hem vist que aquest bloqueig té un impacte significatiu sobre l’expressió de quimiocines directament induïdes per IFN-g, com CXCL9, 10 i 11, tan a nivell de mRNA com de proteïnes, i també té efectes inhibitoris sobre molècules com IL-1b o TNF-a, tot i que aquestes tendències no han arribat a ser estadísticament significatives. L’estimulació de les artèries amb IFN-g recombinant ha resultat en un augment de l’expressió d’aquestes mateixes quimiocines i també dels receptors CCR2 i CXCR3.
L'IFN-g també té un efecte estimulador de l’expressió de quimiocines sobre el component cel·lular majoritari de la paret arterial, les cèl·lules musculars llises (VSMC). L'IFN-g estimula en aquest tipus cel·lular les quimiocines CXCL9, 10 i 11, i CCL2, i també les molècules d’adhesió ICAM-1 i VCAM-1, i els factors de transcripció Stat-1 i Stat-3.
Aquests augments de la síntesi de quimiocines i molècules d'adhesió es tradueixen en un augment de la migració (a través de CXCR3) i de l'adhesió de limfòcits i monòcits cap a les VSMC. El contacte entre aquests tipus cel·lulars ja provoca per si mateix un increment de l'expressió de quimiocines, i aquesta estimulació desapareix a l'inhibir l'IFN-g.
Així doncs, l'IFN-g sembla tenir un paper significatiu en la progressió de l'infiltrat inflamatori a través de la paret arterial. El seu alliberament per part de les cèl·lules inflamatòries provoca en les VSMCs un increment de la síntesi de molècules que provoquen la migració i l'adhesió de les pròpies cèl·lules inflamatòries. Aquests resultats proporcionen les primeres evidencies sobre el
paper funcional del l’IFNg en l’arteritis de cèl·lules gegants i constitueixen un primer pas per explorar el seu potencial com a possible diana terapèutica en l'ACG.GCA is a granulomatous vasculitis of the elderly, affecting large and medium-sized arteries. Glucocorticoids are the cornerstone of GCA treatment, but unfortunately the majority of patients experience undesirable side effects and relapse when glucocorticoids are tapered. This observation underlines the need for searching alternative therapies in GCA. Investigating new treatments requires a better understanding of GCA pathogenesis and the development of functional models for pre-clinical testing, since there are no animal models for GCA.
IFN-g, highly expressed in GCA patients compared to controls, is a cytokine implicated in multiple inflammatory-related pathways, and it may contribute to GCA pathogenesis by participating in lymphocyte Th1 differentiation and by activating macrophages promoting granuloma formation. However, this is extrapolated from its known biologic functions and the functional role of IFN-g in GCA has not been explored.
Our main objectives were: to develop a human temporal artery culture model for functional studies, and to explore IFN-g roles by using a neutralizing, fully human monoclonal antibody against IFN-g.
In our first study we developed a temporal artery culture model in tri-dimensional matrix where viability and morphology is preserved for up to 2 weeks. In this model we have confirmed differences in expression of pro-inflammatory molecules between GCA and control arteries. We have also observed that glucocorticoids inhibit mRNA expression of pro-inflammatory cytokines such as IFN-g and IL-1b or chemokines CCL3, 4 and 5, as well as a reduction in macrophage infiltration assessed by CD68 immunostaining. Interestingly, glucocorticoids did not have a significant effect on the expression of molecules involved in vascular remodelling such as PDGF, TGFb or collagens.
In the second study, we used a fully human neutralizing antibody against IFN-g in order to inhibit IFN-g effects. Treatment of cultured arteries with this antibody reduced the expression and phosphorylation of Stat-1, a pivotal transcription factor in IFN-g-driven canonical signalling pathways. IFN-g blockade led to significant decrease in mRNA and protein expression of
CXCL9, 10 and 11 in cultured arteries and a decrease in infiltrating CD68 macrophages. Stimulating GCA arteries with recombinant IFN-g elicited the opposite effects.
We demonstrated that IFN-g had significant effects on the main cellular component of the arterial wall, vascular smooth muscle cells (VSMC) by inducing chemokine (CXCL9, 10 and 11, and CCL2) and adhesion molecule (ICAM-1 and VCAM-1) expression. This resulted in increased PBMCs migration and adhesion to cultured VSMC and to normal arteries exposed to IFN-g.
In summary, IFN-g synthetized by inflammatory cells, acts on VSMCs via Stat1, to enhance the production and secretion of chemokines and adhesion molecules that regulate trafficking of these leukocytes, perpetuating a loop that contributes to the maintenance and progression of the inflammatory infiltrates in the arterial wall. Our work provides first evidences supporting a functional role of IFNg in GCA, a necessary first step to explore its potential as therapeutic target
miR-146a and miR-146b regulate the expression of ICAM-1 in giant cell arteritis
No full textGiant cell arteritis (GCA) is an inflammatory disease of large/medium-sized arteries. MiRNAs are small, noncoding RNAs that inhibit gene expression at post-transcriptional level. Several miRNAs have been shown to be dysregulated in temporal artery biopsies (TABs) from GCA patients, but their role is unknown. The aims of the present work were: to gain insight into the link between inflammation and miRNA up-regulation in GCA; to identify the role of miR-146a and miR-146b. Primary cultures from TABs were treated with IL-1 beta, IL -6, soluble IL -6R (sIL6R), IL -17, IL -22, IFN gamma, LPS and PolyIC. Correlations between cytokine mRNA and miRNA levels were determined in inflamed TABs. Primary cultures from TABs, human aortic endothelial and smooth muscle cells and ex-vivo TAB sections were transfected with synthetic miR-146a and miR-146b to mimic miRNA activities. Cell viability, target gene expression, cytokine levels in culture supernatants were assayed. Treatment of primary cultures from TABs with IL-1 beta and IL -17 increased miR-146a expression while IL-1 beta, IL-6+sIL6R and IFN gamma increased miR-146b expression. IFN gamma and IL-1 beta mRNA levels correlated with miR-146a/b levels. Following transfection, cell viability decreased only in primary cultures from TABs. Moreover, transfection of miR-146a/b mimics increased ICAM-1 gene expression and production of the soluble form of ICAM-1 by primary cultures from TABs and by ex-vivo TABs. ICAM-1 expression was higher in inflamed than normal TABs and ICAM-1 levels correlated with miR-146a/b levels. Expression of miR-146a and miR-146b in GCA appeared to be driven by inflammatory cytokines (e.g. IL-1 beta, IFN gamma). miR-146a and miR-146b seem responsible for the increase of soluble ICAM-1
On the Low-latency region of best-effort links for delay-sensitive streaming traffic
No full textThis letter analyzes the Low-latency Region (LLR) of a best-effort link (i.e., no traffic differentiation, and first come first serve scheduling) carrying both delay-sensitive (DS) streaming and non-delay-sensitive (NDS) background traffic. Moreover, inside the LLR, we show it exists a proportional fair arrival rate allocation for both the DS and NDS traffic streams. This optimal operating point results from maximizing a simple throughput-delay trade-off that considers the NDS traffic load, and the mean delay of the DS packets. To show how the presented trade-off could be used to allocate NDS traffic in a realistic scenario, we use Google Stadia traffic traces to generate the DS flow. Results from this use-case confirm that the throughput-delay trade-off also works regardless the distribution of the packet arrival and packet service times.The author would like to thank Marc Carrascosa for his contribution in taking the Google Stadia measurements. Also, the author wants to especially thank the anonymous reviewers for their constructive, insightful and challenging comments. This work was supported by WINDMAL PGC2018-099959-B-I00 (MCIU/AEI/FEDER,UE), and SGR017-1188 (AGAUR)
114. Effects of tocilizumab on ex-vivo peripheral blood mononuclear cells from patients with GCA
No full textBackground: Giant –cell arteritis (GCA) is a chronic granulomatous vasculitis affecting individuals aged ≥50 years. Glucocorticoids (GCs) are only effective therapy able to induce disease remission but most patients relapse when GC are tapered and suffer from GC-related toxicity. Interleukin-6 (IL-6) is a pleiotropic cytokine that may play a pivotal role in GCA pathogenesis since its transcripts are increased in vascular lesions and elevated serum levels have been reported in active GCA patients. Recently, blocking IL-6 receptor with tocilizumab (TCZ) has demonstrated effectiveness in reducing GCA flares and sparing GC. However, 40% of patients still relapse when GCs are discontinued, indicating heterogeneity in TCZ-response. Therefore, identifying predictors of response to TCZ is crucial. TCZ strongly reduces systemic symptoand acute-phase reactants but little is known about the effects of TCZ on vascular lesions or peripheral blood mononuclear cells (PBMCs). The aim of this study was to investigate transcriptomic changes induced by IL-6 and TCZ+IL6 on ex-vivo PBMCs from patients in clinical remission.
Methods: PBMCs were isolated from 17 patients in remission and 5 age and sex – matched controls using Lymphoprep™ density gradient. Fourteen patients were receiving prednisone (<5mg/day) and 4 patients were in sustained remission without prednisone. PBMCs were exposed to recombinant human (rh) IL-6 (IL6 group) (20 ng/ml) and combination of rhIL-6 and TCZ (20 µg/ml) (TCZ+IL6 group) and cultured for 24h. Total RNA was extracted from cells using TRIzol™ reagent. RNA (100ng/sample) was processed using nCounter Prep Station screening for 256 genes from Human Inflammation Nanostring panel. Barcode counts were processed with nSolver 4.0 Software. Normalized data were analysed using R Studio 4.0.3 and paired or unpaired Wilcoxon tests were applied.
Results: Total count of PBMCs was significantly lower in GCA compared to controls (1.26±0.43, 2.53±0.2 cell/ml, respectively; p<0.0001). At transcriptomic level, 64 transcripts were differentially expressed between controls and patients, even considered in clinical remission; 5 transcripts were increased and 59 were decreased in GCA. After stimulation with IL-6, 12 transcripts were increased and 34 decreased respect to baseline condition. A total of 113 transcripts were differentially expressed between IL-6 group and TCZ+IL6 group. After correction for multiple comparisons, 73 transcripts remained significant (FDR<0.05), of which 63 transcripts were increased with TCZ exposure. However, 8 transcripts that were increased with IL-6 stimulation, decreased their expression after exposure to TCZ: STAT3, HIF1A, CCL2, CCR1, BCL6, MYC, TLR2 and C3AR1; all of them being target genes of STAT3.
Conclusions: Patients with GCA in clinical remission still have transcriptomic differences with healthy controls. Tocilizumab reduces expression of STAT3 and 7 other transcripts downstream from STAT3, important for differentiation of naïve T helper cells to follicular T helper cells (BCL6), cell growth/apoptosis (MYC), monocyte trafficking and activation (CCL2, CR1), activation of IL-6 (TLR2) and hypoxia (HIF1A). Validation is in process. Studies in active, treatment-naïve patients are ongoing.
Disclosures: Marc Corbera- Bellalta, Farah Kamberovic, Roser Alba-Rovira, Marco A Alba, and Patricia Pérez-Galán have no disclosures to report. Georgina Espigol-Frigolé reports consulting for Janssen and Hoffmann-La Roche; meeting attendance suport from Boehringer Ingelheim. Sergio Prieto-Gonzalez reports lecturing for Roche; meeting attendance support from Italfarmo and CSL Behring. Maria C Cid reports a research grant from Kiniksa; consulting for Janssen, GSK, and Abbvie; educational grant from GSK and Vifor; meeting attendance support from Roche and Kiniksa. Funding received from EU Horizon 2020 research and innovation program (Marie Skłodowska-Curie grant agreement No. 813545), Spanish Ministry for Science and Innovation /AEI project No. PID2020-114909RB-I00, and the Vasculitis Foundation
THU0008 GM-CSF PATHWAY SIGNATURE IDENTIFIED IN TEMPORAL ARTERY BIOPSIES OF PATIENTS WITH GIANT CELL ARTERITIS
No full text
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
No full textEffective 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
