Croce Cristina, Garrido Facundo, Dinamarca Sofía, Santi-Rocca Julien, Marion Sabrina, Blanchard Nicolas, Mayorga Luis S, Cebrian Ignacio
Instituto de Histología y Embriología de Mendoza (IHEM) - Universidad Nacional de Cuyo - CONICET, Mendoza, Argentina.
Institut Toulousain des Maladies Infectieuses et Inflammatoires (Infinity), Inserm/CNRS/Université Toulouse 3, Toulouse, France.
Front Cell Dev Biol. 2022 Mar 4;10:837574. doi: 10.3389/fcell.2022.837574. eCollection 2022.
Dendritic cells are the most powerful antigen-presenting cells of the immune system. They present exogenous antigens associated with Major Histocompatibility Complex (MHC) Class II molecules through the classical pathway to stimulate CD4+ T cells, or with MHC-I to activate CD8+ T lymphocytes through the cross-presentation pathway. DCs represent one of the main cellular targets during infection by . This intracellular parasite incorporates essential nutrients, such as cholesterol, to grow and proliferate inside a highly specialized organelle, the parasitophorous vacuole (PV). While doing so, modulates the host immune response through multiple interactions with proteins and lipids. Cholesterol is an important cellular component that regulates cellular physiology at the structural and functional levels. Although different studies describe the relevance of cholesterol transport for exogenous antigen presentation, the molecular mechanism underlying this process is not defined. Here, we focus our study on the inhibitor U18666A, a drug widely used to arrest multivesicular bodies biogenesis that interrupts cholesterol trafficking and changes the lipid composition of intracellular membranes. Upon bone marrow-derived DC (BMDC) treatment with U18666A, we evidenced a drastic disruption in the ability to present exogenous soluble and particulate antigens to CD4+ and CD8+ T cells. Strikingly, the presentation of -associated antigens and parasite proliferation were hampered in treated cells. However, neither antigen uptake nor BMDC viability was significantly affected by the U18666A treatment. By contrast, this drug altered the transport of MHC-I and MHC-II molecules to the plasma membrane. Since U18666A impairs the formation of MVBs, we analyzed in infected BMDCs the ESCRT machinery responsible for the generation of intraluminal vesicles. We observed that different MVBs markers, including ESCRT proteins, were recruited to the PV. Surprisingly, the main ESCRT-III component CHMP4b was massively recruited to the PV, and its expression level was upregulated upon BMDC infection by . Finally, we demonstrated that BMDC treatment with U18666A interrupted cholesterol delivery and CHMP4b recruitment to the PV, which interfered with an efficient parasite replication. Altogether, our results highlight the importance of cholesterol trafficking and MVBs formation in DCs for optimal antigen presentation and proliferation.
树突状细胞是免疫系统中最强大的抗原呈递细胞。它们通过经典途径呈递与主要组织相容性复合体（MHC）II类分子相关的外源性抗原，以刺激CD4+ T细胞，或通过交叉呈递途径与MHC-I结合，激活CD8+ T淋巴细胞。DCs是感染期间的主要细胞靶点之一。这种细胞内寄生虫摄取胆固醇等必需营养物质，在一个高度特化的细胞器——寄生泡（PV）内生长和增殖。在此过程中，它通过与蛋白质和脂质的多种相互作用来调节宿主免疫反应。胆固醇是一种重要的细胞成分，在结构和功能水平上调节细胞生理。尽管不同的研究描述了胆固醇转运对外源性抗原呈递的相关性，但这一过程的分子机制尚未明确。在这里，我们将研究重点放在抑制剂U18666A上，该药物广泛用于阻止多囊泡体的生物发生，它会中断胆固醇运输并改变细胞内膜的脂质组成。在用U18666A处理骨髓来源的DC（BMDC）后，我们发现其向外源性可溶性和颗粒性抗原呈递给CD4+和CD8+ T细胞的能力受到了严重破坏。令人惊讶的是，处理后的细胞中与寄生虫相关的抗原呈递和寄生虫增殖均受到阻碍。然而，U18666A处理对抗原摄取和BMDC活力均无显著影响。相比之下，这种药物改变了MHC-I和MHC-II分子向质膜的转运。由于U18666A会损害多囊泡体的形成，我们在感染了寄生虫的BMDC中分析了负责腔内小泡生成的ESCRT机制。我们观察到不同的多囊泡体标记物，包括ESCRT蛋白，被募集到寄生泡中。令人惊讶的是，主要的ESCRT-III成分CHMP4b大量被募集到寄生泡中，并且在BMDC被寄生虫感染后其表达水平上调。最后，我们证明用U18666A处理BMDC会中断胆固醇向寄生泡的递送以及CHMP4b向寄生泡的募集，这会干扰寄生虫的有效复制。总之，我们的结果突出了DCs中胆固醇运输和多囊泡体形成对于最佳抗原呈递和寄生虫增殖的重要性。
