**1. Introduction**

Blood–brain barrier (BBB) integrity is necessary to protect the brain from injuries such as toxins and germs, as well as to help in maintaining central nervous system (CNS) homeostasis [1]. BBB activation and dysfunction contributes to several brain pathologies. Many factors are able to induce BBB

dysfunction such as inflammatory mediators, matrix metalloproteinases, free radicals, and vascular endothelial growth factor, among others [2].

Bacteria of the genus *Brucella* produce several types of inflammatory disorders [3]. Neurobrucellosis is a neurodegenerative inflammatory disorder caused by invasion of the CNS by *Brucella* spp. and constitutes the most morbid pathology associated with this infection [4]. One of the most characteristic clinical signs of this disease is pleocytosis; i.e., the presence of leukocytes in the cerebrospinal fluid [4].

We have recently described a putative mechanism employed by *Brucella abortus* to gain access to the CNS. We have demonstrated, using an in vitro model, that *B. abortus* traverses the BBB into the cerebral parenchyma inside infected monocytes, by a mechanism known as "Trojan horse". Moreover, infected monocytes act as bacterial source for de novo infection of glial cells [5]. In addition, we have described that activation of glial cells by *B. abortus* is crucial in neurobrucellosis pathology [6]. *B. abortus*-activated astrocytes and microglia secrete pro-inflammatory mediators such as tumor necrosis factor (TNF)-<sup>α</sup>, interleukin (IL)-6, IL-1β, C-C motif chemokine ligand 2 CCL-2, C-X-C motif chemokine ligand 1 (CXCL1), metalloproteinase (MMP)-9, and nitric oxide (NO), among others [6–8]. These inflammatory mediators, and astrocytes/microglia-secreted IL-1β in particular, activate the BBB, allowing monocyte and neutrophil transmigration [9]. The effect of glial cell activation on BBB cells is well known [9–11]. However, whether peripheral inflammation induced by *Brucella*-activated cells can modify the BBB remains unknown.

Platelets are well characterized as responsible for maintaining vascular integrity in addition to being hemostatic mediators [12]. In the last few years, the immune function of platelets has been described in both homeostasis and pathology [12–14]. Platelets express several immune receptors such as toll-like receptors (TLR) and Fc receptors, which allow the recognition of different pathogens [12]. Upon pathogen recognition, platelets can be activated and secrete a wide variety of immunomodulatory mediators present in their granules [15–17]. The immunoregulatory functions of pathogen-activated platelets have been described recently, as well as their ability to activate endothelial cells, including the microvascular endothelium of the BBB [14,18–20]. We have recently described the interactions between platelets and *B. abortus* [14]. *B. abortus* is able to invade, infect platelets, and activate them. As a consequence of this interaction, platelets establish complexes with *B. abortus*-infected monocytes, increasing the efficiency of the infection and modulating monocyte and neutrophil functions [14,21]. Moreover, we demonstrated that complexes between platelets and both monocytes and neutrophils are more abundant in patients with brucellosis than in healthy donors [21].

We have already described the effect of glial cell activation on BBB cells during neurobrucellosis; however, whether platelets activated by *Brucella* can modify the BBB remains unexplored. Therefore, the aim of this work was to elucidate whether *B. abortus*-activated platelets can activate the BBB and affect transmigration of monocytes and neutrophils. Here, we demonstrated that *B. abortus*-activated platelets activate brain microvascular endothelial cells, and other endothelial cells, through Erk1/2 signaling pathway, leading monocytes and neutrophils to traverse polarized brain microvascular endothelial monolayers.
