Interleukin-6 (IL-6)/IL-6 receptor and persistence of inflammation in Giant Cell Arteritis. Effects of IL-6 receptor blockade with tocilizumab

Abstract

[eng] Giant cell arteritis (GCA) is a chronic granulomatous vasculitis affecting large- and medium-sized vessels. This disease can lead to different symptoms related to vascular or systemic inflammation, such as fever and visual loss, and its exact etiology remains to be elucidated. Current treatment of GCA patients is based on glucocorticoids administration. However, not all patients respond properly to this treatment and the disadvantages associated to glucocorticoids promotes the search for new therapeutic alternatives. Blockade of IL-6 signaling with tocilizumab (TCZ), a humanized monoclonal antibody against IL-6 receptor (IL-6R), represents a newly promising alternative, supported by the results of two recently published clinical trials. However, beyond its implication in the acute phase response, the role of IL-6 in the pathogenesis of GCA and vascular inflammation is still unknown. GCA patients treated with TCZ also showed a decrease of acute-phase proteins, which are usually used to monitor disease activity. Therefore, the utilization of this monoclonal antibody remarks the urgency to find alternative biomarker not directly related with IL-6 signaling to monitor GCA patients treated with TCZ. Considering all this information, the aim of this doctoral thesis was to better understand the role of IL-6 in GCA pathogenesis as well as the impact of IL-6R blockade with TCZ. In addition, we aimed to test the potential of osteopontin (OPN) as a biomarker of disease activity in patients treated with this monoclonal antibody. The results from the present study show that IL-6 and IL-6R are remarkably increased in temporal artery lesions from GCA patients compared with control arteries. Co-culture experiments suggest that vascular smooth muscle cells (VSMC) may be an important source of IL-6. IL-6R was found upregulated in GCA lesions, particularly at the granulomatous areas. Co-culture experiments supported this result since IL-6R protein expression was increased in mononuclear cells when co-cultured with VSMC. Contrary to what was observed in tissue, serum levels of sIL-6R showed no differences between GCA patients and controls. The artery culture model was used to better understand the impact of TCZ. IL-6R blockade resulted in a significant decrease in the mRNA expression of STAT3 and SOCS3 after 5 days in culture. However, phosphorylation levels of STAT3 were not modified by TCZ treatment. Co-culture results suggest that under inflammatory conditions the inhibitory effect of TCZ on STAT3 activation may be partially compensated by alternative mechanisms. IL-6R blockade with TCZ also decreased CCL2 and increased the expression of CXCL9 and CXCL10 in cultured temporal arteries. Based on in vitro results, IL-6R blockade may promote an upregulation of CXCL9 and CXCL10 expression in mononuclear cells, that may explain the increased expression observed in cultured arteries. The upregulation of this chemokines may be due to an increase in STAT1 expression and activation after TCZ treatment. IL-6R blockade with TCZ also induced a reduction in the adhesion and migratory capacity of mononuclear cells. These results suggest that IL-6R blockade with TCZ may contribute to decrease tissue inflammation by limiting the advent of new

inflammatory cells. Further research is needed to better understand the molecules involved in TCZ modulation of these processes. TCZ treatment of cultured arteries did not affect OPN expression in GCA lesions. Consistently, while levels of C-reactive protein (CRP) were virtually undetectable after IL- 6R blockade, serum concentration of OPN was similar in patients on glucocorticoid or TCZ maintained remission. All together, these data suggest that sOPN could be a useful biomarker of disease activity for TCZ treated patients. However, the role of sOPN needs to be further explored in larger studies with longitudinal cohorts.

[cat] L’arteritis de cèl·lules gegants (ACG) és una malaltia inflamatòria crònica d’etiologia desconeguda que afecta les arteries de mitjà i gran calibre. El tractament actual es basa en l’administració de glucocorticoides tot i que presenten efectes adversos i molts pacients experimenten recaigudes. Aquest fet promou la recerca de teràpies alternatives o complementaries. Recentment, s’han publicat els resultats de dos assajos clínics on s’ha vist que un nou fàrmac anomenat tocilizumab (TCZ), un anticòs que bloqueja el receptor de la IL-6 (IL-6R), podria ser una bona alternativa terapèutica per al pacients amb ACG. No obstant, el paper de la IL-6 en la patogènesi de l’ACG és encara desconegut. A més, l’ús del TCZ ha posat de manifest la necessitat de buscar biomarcadors alternatius als clàssicament utilitzats per monitoritzar els pacients, ja que el tractament amb aquest anticòs redueix l’expressió de les proteïnes de fase aguda, les quals són induïdes per la IL-6. Els objectius de la present tesi doctoral han estat per tant, entendre millor el paper de la IL-6 en l’ACG, així com estudiar l’impacte del bloqueig del IL-6R amb TCZ. Tanmateix, s’ha analitzat el potencial de l’osteopontiona (OPN) com a biomarcador alternatiu en pacients tractats amb aquest anticòs. Els resultats del present estudi mostren que tant la IL-6 com el seu receptor es troben incrementats en les lesions de pacients amb ACG. El bloqueig del IL-6R amb TCZ té un efecte clar sobre l’expressió de les quimiocines CCL2, CXCL9 i CXCL10. A més, els resultats suggereixen que el tractament amb TCZ podria contribuir a disminuir la inflamació en els teixits al prevenir l’arribada de noves cèl·lules inflamatòries. En relació al possible paper de l’OPN com a biomarcador en pacients tractats amb TCZ es va veure que els nivells de OPN en sèrum eren similar als dels pacients tractats amb glucocorticoides. Per contra, els nivells de proteïna C reactiva eren pràcticament indetectables en el grup de pacients tractats amb l’anticòs. En conjunt, els resultats mostren que l’OPN podria ser un bon biomarcador de l’activitat de la malaltia en pacients tractats amb TCZ.