Role of Mfn2 in Macrophage Inflammatory Responses

Abstract

[eng] Mitochondria are well known for their role as bioenergetic and biosynthetic organelles. Recently, they also have emerged as one of the main regulators of innate immune responses, mostly for its ability to modulate several signaling pathways through mechanisms such ROS production. Mitofusin-2 (Mfn2) is a GTPase located in the external mitochondrial and ER membranes. It is responsible for the fusion between mitochondria and the ER-mitochondria contacts, both necessary for the correct functioning of both organelles. Even that, the role of Mfn2 in immune responses. Here we demonstrate that Mfn2 is crucial for the pro-inflammatory activation of macrophages. Murine Mfn2-/- macrophages show a disruption in the mitochondrial network morphology that leads to loss in the mitochondrial membrane potential and in mitochondrial respiration. These two defects in the mitochondrial function do not affect their ability to normally generate ATP. However, the production of ROS in the mitochondria from Mfn2-/- macrophages is markedly decreased. This defect in ROS generation leads to a severe dysfunction in macrophage responses, particularly in the inflammatory activation, phagocytosis, and the processing of proteins. First, the decrease in ROS in Mfn2-/- macrophages results in a defective activation of p38, ERK, and NF-kB signaling pathways in response to LPS. The decrease in these signaling cascades leads to a reduction in the production of pro- inflammatory cytokines, severely impairing their ability to undergo pro- inflammatory activation. Second, in addition to show reduced ROS levels, Mfn2-/- also show an accumulation of autophagosomes due to a Mfn2-dependent defect in the autophagosome-lysosome fusion. As a result to both increased autophagy and decreased ROS levels, Mfn2-/- macrophages show decreased expression of type- A scavenger receptors. We demonstrate that these alterations lead to a widespread defect in the phagocytic capabilities of Mfn2-/- macrophages, showing defective phagocytosis of bacteria (both gram positive and gram negative) and apoptotic bodies. Thirdly, Mfn2-/- macrophages also show a defect in the bactericidal activity of phagocyted bacteria, as well as a defective proteolysis, being unable to process antigens to present them to CD4+ cells in a MHC-II context, and therefore, potentially impairing their ability to initiate adaptive immune responses. Finally, we demonstrated that Mfn2 is relevant in in vivo models of inflammation or infection. Myeloid-conditional Mfn2-/- mice were infected with either Listeria monocytogenes or Mycobacterium tuberculosis. In both models, Mfn2-/- mice showed a severe decrease in their survival, when compared to their WT counterparts. Furthermore, the colony counts in selected organs (spleen and liver for listeria, spleen and lung for tuberculosis) was significantly increased in Mfn2-/- mice, indicating that Mfn2 in macrophages is required to effectively control bacterial infections. In addition, we performed a model of sterile inflammation using the irritant DNFB on the mice’s ear. Confirming the in vitro results, Mfn2-/- mice show decreased inflammation in the ear, as confirmed by the decrease in size and weight, and the reduced expression of inflammatory cytokines. All these findings suggest that Mfn2 is a crucial regulator of macrophage pro- inflammatory responses, including production of pro-inflammatory cytokines, phagocytosis, and antigen presentation, through modulation of mitochondrial ROS production, autophagy, and protein processing.

[cat] Apart del seu rol en la regulació del metabolisme, és cada cop més acceptat els mitocondris són un dels principals controladors de les respostes immunes. En aquesta tesi ens centrem en l’estudi de Mitofusina 2 (Mfn2), una GTPasa que es troba a la membrana mitocondrial externa i que promou la fusió entre mitocondris. Degut a que Mfn2 controla aspectes clau de la fisiologia mitocondrial com la respiració, la producció de ROS i l’apoptosi, tots ells intrínsecament lligats en el funcionament de les respostes immunes, decidim estudiar el paper que juga aquesta proteïna en els macròfags, cèl·lules clau en la inflamació. Els macròfags Mfn2-/- presenten una xarxa mitocondrial completament fragmentada, així com una pèrdua del potencial de membrana mitocondrial i una disminució de la respiració. Això fa que les mitocòndries d’aquestes cèl·lules, tot i generar ATP de forma normal, siguin incapaces de produir nivells fisiològics de ROS. Aquesta disminució de ROS provoca que vies de senyalització com ERK, p38 i NF-κB no puguin ser activades per LPS, portant els macròfags Mfn2-/- a ser incapaços de sintetitzar citocines inflamatòries com el TNF-α o la IL-1β, i per tant de generar una resposta inflamatòria eficient. Per altra banda, la deficiència de Mfn2 provoca una acumulació d’autofagosomes en els macròfags. Això genera per una banda un increment de l’apoptosi en aquestes cèl·lules, i per altra un defecte en la seva capacitat fagocítica. Tot això a més és combina amb un defecte en la degradació de bacteris fagocitats i el processament de proteïnes necessari per la presentació antigènic, probablement degut també a la falta de ROS. Finalment demostrem que Mfn2 és crucial en les respostes inflamatòries usant 3 models in vivo. En dos d’ells mostrem com els macròfags necessiten Mfn2 per controlar les infeccions de listèria i tuberculosis, mentre que el l’últim demostrem que Mfn2 també és necessària en la inflamació asèptica. Per concloure, en aquesta tesi demostrem que la Mfn2 és crucial per l’activació pro- inflamatòria del macròfag, afectant tres processos clau: la producció de citocines inflamatòries, la fagocitosi i la presentació antigènica.