Platelets promote human macrophages-mediated macropinocytosis of Clostridioides difficile.

Clostridioides difficile is the main causative agent of hospital-acquired diarrhea and the potentially lethal disease, C. difficile infection. The cornerstone of the current therapy is the use of antibiotics, which is not fully effective. The molecular mechanisms, inflammatory conditions and host-immune responses that could benefit the persistence or elimination of C. difficile remain unclear. Macrophages perform different ways of endocytosis as part of their immune surveillance functions and platelets, classically known for their coagulatory role, are also important modulators of the immune system. The aim of this study was to evaluate the endocytosis of vegetative C. difficile by human macrophages and the involvement of platelets in this process. Our results showed that both macrophages and platelets interact with live and heat-killed C. difficile. Furthermore, platelets form complexes with human monocytes in healthy donor's fresh blood and the presence of C. difficile increased these cell-cell interactions. Using flow cytometry and confocal microscopy, we show that macrophages can internalize C. difficile and that platelets improve this uptake. By using inhibitors of different endocytic pathways, we demonstrate that macropinocytosis is the route of entry of C. difficile into the cell. Taken together, our findings are the first evidence for the internalization of vegetative non-toxigenic and hypervirulent C. difficile by human macrophages and highlight the role of platelets in innate immunity during C. difficile infection. Deciphering the crosstalk of C. difficile with immune cells could provide new tools for understanding the pathogenesis of C. difficile infection and for the development of host-directed therapies.

Brucella abortus RNA does not polarize macrophages to a particular profile but interferes with M1 polarization.

Monocytes and macrophages play a central role in chronic brucellosis. Brucella abortus (Ba) is an intracellular pathogen that survives inside these cells. On the other hand, macrophages could be differentiated into classical (M1), alternative (M2) or other less-identified profiles. We have previously shown that Ba RNA (a bacterial viability-associated PAMP or vita-PAMP) is a key molecule by which Ba can evade the host immune response. However, we did not know if macrophages could be polarized by this vita-PAMP. To assess this, we used two different approaches: we evaluated if Ba RNA per se was able to differentiate macrophages to M1 or M2 or, given that Ba survives inside macrophages once a Th1 response is established (i.e., in the presence of IFN-γ), we also analysed if Ba RNA could interfere with M1 polarization. We found that Ba RNA alone does not polarize to M1 or M2 but activates human macrophages instead. However, our results show that Ba RNA does interfere with M1 polarization while they are being differentiated. This vita-PAMP diminished the M1-induced CD64, and MHC-II surface expression on macrophages at 48 h. This phenomenon was not associated with an alternative activation of these cells (M2), as shown by unchanged CD206, DC-SIGN and CD163 surface expression. When evaluating glucose metabolism, we found that Ba RNA did not modify M1 glucose consumption or lactate production. However, production of Nitrogen Reactive Species (NRS) did diminish in Ba RNA-treated M1 macrophages. Overall, our results show that Ba RNA could alter the proper immune response set to counterattack the bacteria that could persist in the host establishing a chronic infection.

SLAMF1 signaling induces Mycobacterium tuberculosis uptake leading to endolysosomal maturation in human macrophages.

Tuberculosis dates back to ancient times but it is not a problem of the past. Each year, millions of people die from tuberculosis. After inhalation of infectious droplet nuclei, Mycobacterium tuberculosis reaches the lungs where it can manipulate the immune system and survive within host macrophages, establishing a persistent infection. The signaling lymphocytic activation molecule family member 1 (SLAMF1) is a self-ligand receptor that can internalize gram-negative bacteria and regulate macrophages' phagosomal functions. In tuberculosis, SLAMF1 promotes Th1-protective responses. In this work, we studied the role of SLAMF1 on macrophages' functions during M. tuberculosis infection. Our results showed that both M. tuberculosis and IFN-γ stimulation induce SLAMF1 expression in macrophages from healthy donor and Tohoku Hospital Pediatrcs-1 cells. Costimulation through SLAMF1 with an agonistic antibody resulted in an enhanced internalization of M. tuberculosis by macrophages. Interestingly, we found that SLAMF1 interacts with M. tuberculosis and colocalizes with the bacteria and with early and late endosomes/lysosomes markers (EEA1 and LAMP2), suggesting that SLAMF1 recognize M. tuberculosis and participate in the endolysosomal maturation process. Notably, increased levels of SLAMF1 were detected in CD14 cells from pleural effusions of tuberculosis patients, indicating that SLAMF1 might have an active function at the site of infection. Taken together, our results provide evidence that SLAMF1 improves the uptake of M. tuberculosis by human monocyte-derived macrophages.

Fatty acid oxidation of alternatively activated macrophages prevents foam cell formation, but Mycobacterium tuberculosis counteracts this process via HIF-1α activation.

The ability of Mycobacterium tuberculosis (Mtb) to persist inside host cells relies on metabolic adaptation, like the accumulation of lipid bodies (LBs) in the so-called foamy macrophages (FM), which are favorable to Mtb. The activation state of macrophages is tightly associated to different metabolic pathways, such as lipid metabolism, but whether differentiation towards FM differs between the macrophage activation profiles remains unclear. Here, we aimed to elucidate whether distinct macrophage activation states exposed to a tuberculosis-associated microenvironment or directly infected with Mtb can form FM. We showed that the triggering of signal transducer and activator of transcription 6 (STAT6) in interleukin (IL)-4-activated human macrophages (M(IL-4)) prevents FM formation induced by pleural effusion from patients with tuberculosis. In these cells, LBs are disrupted by lipolysis, and the released fatty acids enter the ß-oxidation (FAO) pathway fueling the generation of ATP in mitochondria. Accordingly, murine alveolar macrophages, which exhibit a predominant FAO metabolism, are less prone to become FM than bone marrow derived-macrophages. Interestingly, direct infection of M(IL-4) macrophages with Mtb results in the establishment of aerobic glycolytic pathway and FM formation, which could be prevented by FAO activation or inhibition of the hypoxia-inducible factor 1-alpha (HIF-1α)-induced glycolytic pathway. In conclusion, our results demonstrate that Mtb has a remarkable capacity to induce FM formation through the rewiring of metabolic pathways in human macrophages, including the STAT6-driven alternatively activated program. This study provides key insights into macrophage metabolism and pathogen subversion strategies.

Brucella abortus-Stimulated Platelets Activate Brain Microvascular Endothelial Cells Increasing Cell Transmigration through the Erk1/2 Pathway.

Central nervous system invasion by bacteria of the genus Brucella results in an inflammatory disorder called neurobrucellosis. A common feature associated with this pathology is blood-brain barrier (BBB) activation. However, the underlying mechanisms involved with such BBB activation remain unknown. The aim of this work was to investigate the role of Brucella abortus-stimulated platelets on human brain microvascular endothelial cell (HBMEC) activation. Platelets enhanced HBMEC activation in response to B. abortus infection. Furthermore, supernatants from B. abortus-stimulated platelets also activated brain endothelial cells, inducing increased secretion of IL-6, IL-8, CCL-2 as well as ICAM-1 and CD40 upregulation on HBMEC compared with supernatants from unstimulated platelets. Outer membrane protein 19, a B. abortus lipoprotein, recapitulated B. abortus-mediated activation of HBMECs by platelets. In addition, supernatants from B. abortus-activated platelets promoted transendothelial migration of neutrophils and monocytes. Finally, using a pharmacological inhibitor, we demonstrated that the Erk1/2 pathway is involved in the endothelial activation induced by B. abortus-stimulated platelets and also in transendothelial migration of neutrophils. These results describe a mechanism whereby B. abortus-stimulated platelets induce endothelial cell activation, promoting neutrophils and monocytes to traverse the BBB probably contributing to the inflammatory pathology of neurobrucellosis.

Brucella abortus-infected platelets modulate the activation of neutrophils.

Brucellosis is a contagious disease caused by bacteria of the genus Brucella. Platelets (PLTs) have been widely involved in the modulation of the immune response. We have previously reported the modulation of Brucella abortus-mediated infection of monocytes. As a result, PLTs cooperate with monocytes and increase their inflammatory capacity, promoting the resolution of the infection. Extending these results, in this study we demonstrate that patients with brucellosis present slightly elevated levels of complexes between PLTs and both monocytes and neutrophils. We then assessed whether PLTs were capable of modulating functional aspects of neutrophils. The presence of PLTs throughout neutrophil infection increased the production of interleukin-8, CD11b surface expression and reactive oxygen species formation, whereas it decreased the expression of CD62L, indicating an activated status of these cells. We next analyzed whether this modulation was mediated by released factors. To discriminate between these options, neutrophils were treated with supernatants collected from B. abortus-infected PLTs. Our results show that CD11b expression was induced by soluble factors of PLTs but direct contact between cell populations was needed to enhance the respiratory burst. Additionally, B. abortus-infected PLTs recruit polymorphonuclear (PMN) cells to the site of infection. Finally, the presence of PLTs did not modify the initial invasion of PMN cells by B. abortus but improved the control of the infection at extended times. Altogether, our results demonstrate that PLTs interact with neutrophils and promote a proinflammatory phenotype which could also contribute to the resolution of the infection.

Bacterial RNA Contributes to the Down-Modulation of MHC-II Expression on Monocytes/Macrophages Diminishing CD4+ T Cell Responses.

Brucella abortus, the causative agent of brucellosis, displays many resources to evade T cell responses conducive to persist inside the host. Our laboratory has previously showed that infection of human monocytes with B. abortus down-modulates the IFN-γ-induced MHC-II expression. Brucella outer membrane lipoproteins are structural components involved in this phenomenon. Moreover, IL-6 is the soluble factor that mediated MHC-II down-regulation. Yet, the MHC-II down-regulation exerted by lipoproteins was less marked than the one observed as consequence of infection. This led us to postulate that there should be other components associated with viable bacteria that may act together with lipoproteins in order to diminish MHC-II. Our group has recently demonstrated that B. abortus RNA (PAMP related to pathogens' viability or vita-PAMP) is involved in MHC-I down-regulation. Therefore, in this study we investigated if B. abortus RNA could be contributing to the down-regulation of MHC-II. This PAMP significantly down-modulated the IFN-γ-induced MHC-II surface expression on THP-1 cells as well as in primary human monocytes and murine bone marrow macrophages. The expression of other molecules up-regulated by IFN-γ (such as co-stimulatory molecules) was stimulated on monocytes treated with B. abortus RNA. This result shows that this PAMP does not alter all IFN-γ-induced molecules globally. We also showed that other bacterial and parasitic RNAs caused MHC-II surface expression down-modulation indicating that this phenomenon is not restricted to B. abortus. Moreover, completely degraded RNA was also able to reproduce the phenomenon. MHC-II down-regulation on monocytes treated with RNA and L-Omp19 (a prototypical lipoprotein of B. abortus) was more pronounced than in monocytes stimulated with both components separately. We also demonstrated that B. abortus RNA along with its lipoproteins decrease MHC-II surface expression predominantly by a mechanism of inhibition of MHC-II expression. Regarding the signaling pathway, we demonstrated that IL-6 is a soluble factor implicated in B. abortus RNA and lipoproteins-triggered MHC-II surface down-regulation. Finally, CD4+ T cells functionality was affected as macrophages treated with these components showed lower antigen presentation capacity. Therefore, B. abortus RNA and lipoproteins are two PAMPs that contribute to MHC-II down-regulation on monocytes/macrophages diminishing CD4+ T cell responses.

Inhibition of antigen presentation by Brucella: many more than many ways.

Brucella infection activates the immune system and favors the differentiation of CD4+ and CD8+ T cells. To persist during a long time inside macrophages evading immune surveillance of these T cells the pathogen must exploit different evasion strategies. We review the mechanisms whereby Brucella, through TLR signaling, inhibits MHC class I and II antigen presentation, allowing infected macrophages to become effective niches for Brucella survival.

Brucella abortus Traverses Brain Microvascular Endothelial Cells Using Infected Monocytes as a Trojan Horse.

Neurobrucellosis is an inflammatory disease caused by the invasion of Brucella spp. to the central nervous system (CNS). The pathogenesis of the disease is not well characterized; however, for Brucella to gain access to the brain parenchyma, traversing of the blood-brain barrier (BBB) must take place. To understand the CNS determinants of the pathogenesis of B. abortus, we have used the in vitro BBB model of human brain microvascular endothelial cells (HBMEC) to study the interactions between B. abortus and brain endothelial cells. In this study, we showed that B. abortus is able to adhere and invade HBMEC which was dependent on microtubules, microfilaments, endosome acidification and de novo protein synthesis. After infection, B. abortus rapidly escapes the endosomal compartment of HBMEC and forms a replicative Brucella-containing vacuole that involves interactions with the endoplasmic reticulum. Despite the ability of B. abortus to invade and replicate in HBMEC, the bacterium was unable by itself to traverse HBMEC, but could traverse polarized HBMEC monolayers within infected monocytes. Importantly, infected monocytes that traversed the HBMEC monolayer were a bacterial source for de novo infection of glial cells. This is the first demonstration of the mechanism whereby B. abortus is able to traverse the BBB and infect cells of the CNS. These results may have important implications in our understanding of the pathogenesis of neurobrucellosis.

Platelets Promote Brucella abortus Monocyte Invasion by Establishing Complexes With Monocytes.

Brucellosis is an infectious disease elicited by bacteria of the genus Brucella. Platelets have been extensively described as mediators of hemostasis and responsible for maintaining vascular integrity. Nevertheless, they have been recently involved in the modulation of innate and adaptive immune responses. Although many interactions have been described between Brucella abortus and monocytes/macrophages, the role of platelets during monocyte/macrophage infection by these bacteria remained unknown. The aim of this study was to investigate the role of platelets in the immune response against B. abortus. We first focused on the possible interactions between B. abortus and platelets. Bacteria were able to directly interact with platelets. Moreover, this interaction triggered platelet activation, measured as fibrinogen binding and P-selectin expression. We further investigated whether platelets were involved in Brucella-mediated monocyte/macrophage early infection. The presence of platelets promoted the invasion of monocytes/macrophages by B. abortus. Moreover, platelets established complexes with infected monocytes/macrophages as a result of a carrier function elicited by platelets. We also evaluated the ability of platelets to modulate functional aspects of monocytes in the context of the infection. The presence of platelets during monocyte infection enhanced IL-1ß, TNF-α, IL-8, and MCP-1 secretion while it inhibited the secretion of IL-10. At the same time, platelets increased the expression of CD54 (ICAM-1) and CD40. Furthermore, we showed that soluble factors released by B. abortus-activated platelets, such as soluble CD40L, platelet factor 4, platelet-activating factor, and thromboxane A2, were involved in CD54 induction. Overall, our results indicate that platelets can directly sense and react to B. abortus presence and modulate B. abortus-mediated infection of monocytes/macrophages increasing their pro-inflammatory capacity, which could promote the resolution of the infection.

Formation of Foamy Macrophages by Tuberculous Pleural Effusions Is Triggered by the Interleukin-10/Signal Transducer and Activator of Transcription 3 Axis through ACAT Upregulation.

The ability of Mycobacterium tuberculosis (Mtb) to persist in its human host relies on numerous immune evasion strategies, such as the deregulation of the lipid metabolism leading to the formation of foamy macrophages (FM). Yet, the specific host factors leading to the foamy phenotype of Mtb-infected macrophages remain unknown. Herein, we aimed to address whether host cytokines contribute to FM formation in the context of Mtb infection. Our approach is based on the use of an acellular fraction of tuberculous pleural effusions (TB-PE) as a physiological source of local factors released during Mtb infection. We found that TB-PE induced FM differentiation as observed by the increase in lipid bodies, intracellular cholesterol, and expression of the scavenger receptor CD36, as well as the enzyme acyl CoA:cholesterol acyl transferase (ACAT). Importantly, interleukin-10 (IL-10) depletion from TB-PE prevented the augmentation of all these parameters. Moreover, we observed a positive correlation between the levels of IL-10 and the number of lipid-laden CD14+ cells among the pleural cells in TB patients, demonstrating that FM differentiation occurs within the pleural environment. Downstream of IL-10 signaling, we noticed that the transcription factor signal transducer and activator of transcription 3 was activated by TB-PE, and its chemical inhibition prevented the accumulation of lipid bodies and ACAT expression in macrophages. In terms of the host immune response, TB-PE-treated macrophages displayed immunosuppressive properties and bore higher bacillary loads. Finally, we confirmed our results using bone marrow-derived macrophage from IL-10-/- mice demonstrating that IL-10 deficiency partially prevented foamy phenotype induction after Mtb lipids exposure. In conclusion, our results evidence a role of IL-10 in promoting the differentiation of FM in the context of Mtb infection, contributing to our understanding of how alterations of the host metabolic factors may favor pathogen persistence.

B. abortus RNA is the component involved in the down-modulation of MHC-I expression on human monocytes via TLR8 and the EGFR pathway.

Despite eliciting a potent CD8+ T cell response, Brucella abortus is able to persist and establish a chronic infection inside its host. We have previously reported that the infection of human monocytes/macrophages with B. abortus inhibits the IFN-γ-induced MHC-I cell surface expression down-modulating cytotoxic CD8+ T cell responses. MHC-I down-modulation depends on bacterial viability and results from the capacity of B. abortus to retain the MHC-I molecules within the Golgi apparatus. Furthermore, we recently demonstrated that epidermal growth factor receptor (EGFR) pathway is involved in this phenomenon and that this is an early event during infection. However, the components and mechanisms whereby B. abortus is able to down-modulate MHC-I remained to be elucidated. In this study we demonstrated that the down-modulation of MHC-I expression is not mediated by well-known Brucella virulence factors but instead by B. abortus RNA, a PAMP associated to viability (vita-PAMP). Surprisingly, completely degraded RNA was also able to inhibit MHC-I expression to the same extent as intact RNA. Accordingly, B. abortus RNA and its degradation products were able to mimic the MHC-I intracellular retention within the Golgi apparatus observed upon infection. We further demonstrated that TLR8, a single-stranded RNA and RNA degradation products sensor, was involved in MHC-I inhibition. On the other hand, neutralization of the EGFR reversed the MHC-I inhibition, suggesting a connection between the TLR8 and EGFR pathways. Finally, B. abortus RNA-treated macrophages display diminished capacity of antigen presentation to CD8+ T cells. Overall, our results indicate that the vita-PAMP RNA as well as its degradation products constitute novel virulence factors whereby B. abortus, by a TLR8-dependent mechanism and through the EGFR pathway, inhibits the IFN-γ-induced MHC-I surface expression on human monocytes/macrophages. Thus, bacteria can hide within infected cells and avoid the immunological surveillance of cytotoxic CD8+ T cells.

Prokaryotic RNA Associated to Bacterial Viability Induces Polymorphonuclear Neutrophil Activation.

Polymorphonuclear neutrophils (PMN) are the first cellular line of antibacterial host defense. They sense pathogens through recognition of pathogen-associated molecular patterns (PAMPs) by innate pattern recognition receptors, such as Toll-like receptors (TLR). The aim of this study was to investigate whether PMN sense bacterial viability and explore which viability factor could be involved in this phenomenon. For this purpose, different functions were evaluated in isolated human PMN using live Escherichia coli (Ec) and heat-killed Ec (HK-Ec). We found that bacterial viability was indispensable to induce PMN activation, as measured by forward-scatter (FSC) increase, CD11b surface expression, chemotaxis, reactive oxygen species (ROS) generation and neutrophil extracellular trap (NET) formation. As uncapped non-polyadenylated prokaryotic mRNA has been recognized as a PAMP associated to bacterial viability by macrophages and dendritic cells, total prokaryotic RNA (pRNA) from live Ec was purified and used as a stimulus for PMN. pRNA triggered similar responses to those observed with live bacteria. No RNA could be isolated from HK-Ec, explaining the lack of effect of dead bacteria. Moreover, the supernatant of dead bacteria was able to induce PMN activation, and this was associated with the presence of pRNA in this supernatant, which is released in the killing process. The induction of bactericidal functions (ROS and NETosis) by pRNA were abolished when the supernatant of dead bacteria or isolated pRNA were treated with RNAse. Moreover, endocytosis was necessary for pRNA-induced ROS generation and NETosis, and priming was required for the induction of pRNA-induced ROS in whole blood. However, responses related to movement and degranulation (FSC increase, CD11b up-regulation, and chemotaxis) were still triggered when pRNA was digested with RNase, and were not dependent on pRNA endocytosis or PMN priming. In conclusion, our results indicate that PMN sense live bacteria through recognition of pRNA, and this sensing triggers potent bactericidal mechanisms.

Brucella abortus-activated microglia induce neuronal death through primary phagocytosis.

Inflammation has long been implicated as a contributor to pathogenesis in neurobrucellosis. Many of the associated neurocognitive symptoms of neurobrucellosis may be the result of neuronal dysfunction resulting from the inflammatory response induced by Brucella abortus infection in the central nervous system. In this manuscript, we describe an immune mechanism for inflammatory activation of microglia that leads to neuronal death upon B. abortus infection. B. abortus was unable to infect or harm primary cultures of mouse neurons. However, when neurons were co-cultured with microglia and infected with B. abortus significant neuronal loss occurred. This phenomenon was dependent on TLR2 activation by Brucella lipoproteins. Neuronal death was not due to apoptosis, but it was dependent on the microglial release of nitric oxide (NO). B. abortus infection stimulated microglial proliferation, phagocytic activity and engulfment of neurons. NO secreted by B. abortus-activated microglia induced neuronal exposure of the "eat-me" signal phosphatidylserine (PS). Blocking of PS-binding to protein milk fat globule epidermal growth factor-8 (MFG-E8) or microglial vitronectin receptor-MFG-E8 interaction was sufficient to prevent neuronal loss by inhibiting microglial phagocytosis without affecting their activation. Taken together, our results indicate that B. abortus is not directly toxic to neurons; rather, these cells become distressed and are killed by phagocytosis in the inflammatory surroundings generated by infected microglia. Neuronal loss induced by B. abortus-activated microglia may explain, in part, the neurological deficits observed during neurobrucellosis.

Brucella abortus down-regulates MHC class II by the IL-6-dependent inhibition of CIITA through the downmodulation of IFN regulatory factor-1 (IRF-1).

Brucella abortus is an intracellular pathogen capable of surviving inside of macrophages. The success of B. abortus as a chronic pathogen relies on its ability to orchestrate different strategies to evade the adaptive CD4+ T cell responses that it elicits. Previously, we demonstrated that B. abortus inhibits the IFN-γ-induced surface expression of MHC class II (MHC-II) molecules on human monocytes, and this phenomenon correlated with a reduction in antigen presentation. However, the molecular mechanisms, whereby B. abortus is able to down-regulate the expression of MHC-II, remained to be elucidated. In this study, we demonstrated that B. abortus infection inhibits the IFN-γ-induced transcription of MHC-II, transactivator (CIITA) and MHC-II genes. Accordingly, we observed that the synthesis of MHC-II proteins was also diminished. B. abortus was not only able to reduce the expression of mature MHC-II, but it also inhibited the expression of invariant chain (Ii)-associated immature MHC-II molecules. Outer membrane protein 19 (Omp19), a prototypical B. abortus lipoprotein, diminished the expression of MHC-II and CIITA transcripts to the same extent as B. abortus infection. IL-6 contributes to these down-regulatory phenomena. In addition, B. abortus and its lipoproteins, through IL-6 secretion, induced the transcription of the negative regulators of IFN-γ signaling, suppressor of cytokine signaling (SOCS)-1 and -3, without interfering with STAT1 activation. Yet, B. abortus lipoproteins via IL-6 inhibit the expression of IFN regulatory factor 1 (IRF-1), a critical regulatory transcription factor for CIITA induction. Overall, these results indicate that B. abortus inhibits the expression of MHC-II molecules at very early points in their synthesis and in this way, may prevent recognition by T cells establishing a chronic infection.

Inhibition of MHC-I by Brucella abortus is an early event during infection and involves EGFR pathway.

Brucella abortus is able to persist inside the host despite the development of potent CD8+ T-cell responses. We have recently reported the ability of B. abortus to inhibit the interferon-γ-induced major histocompatibility complex (MHC)-I cell surface expression on human monocytes. This phenomenon was due to the B. abortus-mediated retention of MHC-I molecules within the Golgi apparatus and was dependent on bacterial viability. However, the implications of bacterial virulence or replicative capacity and the signaling pathways remained unknown. Here we demonstrated that the B. abortus mutant strains RB51 and virB10- are able to inhibit MHC-I expression in the same manner as wild-type B. abortus, even though they are unable to persist inside human monocytes for a long period of time. Consistent with this, the phenomenon was triggered early in time and could be observed at 8 h postinfection. At 24 and 48 h, it was even stronger. Regarding the signaling pathway, targeting epidermal growth factor (EGF) receptor (EGFR), ErbB2 (HER2) or inhibition of tumor necrosis factor-α-converting enzyme, one of the enzymes which generates soluble EGF-like ligands, resulted in partial recovery of MHC-I surface expression. Moreover, recombinant EGF and transforming growth factor-α as well as the combination of both were also able to reproduce the B. abortus-induced MHC-I downmodulation. Finally, when infection was performed in the presence of an extracellular signal-regulated kinase 1/2 (Erk1/2) inhibitor, MHC-I surface expression was significantly recovered. Overall, these results describe how B. abortus evades CD8+ T-cell responses early during infection and exploits the EGFR-ERK signaling pathway to escape from the immune system and favor chronicity.

A Brucella spp. Protease Inhibitor Limits Antigen Lysosomal Proteolysis, Increases Cross-Presentation, and Enhances CD8+ T Cell Responses.

In this study, we demonstrate that the unlipidated (U) outer membrane protein (Omp) 19 from Brucella spp. is a competitive inhibitor of human cathepsin L. U-Omp19 inhibits lysosome cathepsins and APC-derived microsome activity in vitro and partially inhibits lysosomal cathepsin L activity within live APCs. Codelivery of U-Omp19 with the Ag can reduce intracellular Ag digestion and increases Ag half-life in dendritic cells (DCs). U-Omp19 retains the Ag in Lamp-2(+) compartments after its internalization and promotes a sustained expression of MHC class I/peptide complexes in the cell surface of DCs. Consequently, U-Omp19 enhances Ag cross-presentation by DCs to CD8(+) T cells. U-Omp19 s.c. delivery induces the recruitment of CD11c(+)CD8α(+) DCs and monocytes to lymph nodes whereas it partially limits in vivo Ag proteolysis inside DCs. Accordingly, this protein is able to induce CD8(+) T cell responses in vivo against codelivered Ag. Antitumor responses were elicited after U-Omp19 coadministration, increasing survival of mice in a murine melanoma challenge model. Collectively, these results indicate that a cysteine protease inhibitor from bacterial origin could be a suitable component of vaccine formulations against tumors.

Glial Cell-Elicited Activation of Brain Microvasculature in Response to Brucella abortus Infection Requires ASC Inflammasome-Dependent IL-1ß Production.

Blood-brain barrier activation and/or dysfunction are a common feature of human neurobrucellosis, but the underlying pathogenic mechanisms are largely unknown. In this article, we describe an immune mechanism for inflammatory activation of human brain microvascular endothelial cells (HBMEC) in response to infection with Brucella abortus Infection of HBMEC with B. abortus induced the secretion of IL-6, IL-8, and MCP-1, and the upregulation of CD54 (ICAM-1), consistent with a state of activation. Culture supernatants (CS) from glial cells (astrocytes and microglia) infected with B. abortus also induced activation of HBMEC, but to a greater extent. Although B. abortus-infected glial cells secreted IL-1ß and TNF-α, activation of HBMEC was dependent on IL-1ß because CS from B. abortus-infected astrocytes and microglia deficient in caspase-1 and apoptosis-associated speck-like protein containing a CARD failed to induce HBMEC activation. Consistently, treatment of CS with neutralizing anti-IL-1ß inhibited HBMEC activation. Both absent in melanoma 2 and Nod-like receptor containing a pyrin domain 3 are partially required for caspase-1 activation and IL-1ß secretion, suggesting that multiple apoptosis-associated speck-like protein containing CARD-dependent inflammasomes contribute to IL-1ß-induced activation of the brain microvasculature. Inflammasome-mediated IL-1ß secretion in glial cells depends on TLR2 and MyD88 adapter-like/TIRAP. Finally, neutrophil and monocyte migration across HBMEC monolayers was increased by CS from Brucella-infected glial cells in an IL-1ß-dependent fashion, and the infiltration of neutrophils into the brain parenchyma upon intracranial injection of B. abortus was diminished in the absence of Nod-like receptor containing a pyrin domain 3 and absent in melanoma 2. Our results indicate that innate immunity of the CNS set in motion by B. abortus contributes to the activation of the blood-brain barrier in neurobrucellosis and IL-1ß mediates this phenomenon.

A bacterial protease inhibitor protects antigens delivered in oral vaccines from digestion while triggering specific mucosal immune responses.

We report here that a bacterial protease inhibitor from Brucella spp. called U-Omp19 behaves as an ideal constituent for a vaccine formulation against infectious diseases. When co-administered orally with an antigen (Ag), U-Omp19: i) can bypass the harsh environment of the gastrointestinal tract by inhibiting stomach and intestine proteases and consequently increases the half-life of the co-administered Ag at immune inductive sites: Peyer's patches and mesenteric lymph nodes while ii) it induces the recruitment and activation of antigen presenting cells (APCs) and increases the amount of intracellular Ag inside APCs. Therefore, mucosal as well as systemic Ag-specific immune responses, antibodies, Th1, Th17 and CD8(+) T cells are enhanced when U-Omp19 is co-administered with the Ag orally. Finally, this bacterial protease inhibitor in an oral vaccine formulation confers mucosal protection and reduces parasite loads after oral challenge with virulent Toxoplasma gondii.