Background: The cross-talk between the host and its microbiota plays a key role in the promotion of health. The production of metabolites such as polyamines by intestinal-resident bacteria is part of this symbiosis shaping host immunity. The polyamines putrescine, spermine, and spermidine are abundant within the gastrointestinal tract and might substantially contribute to gut immunity.Objective: We aimed to characterize the polyamine spermidine as a modulator of T-cell differentiation and function. Methods: Naive T cells were isolated from wild-type mice or cord blood from healthy donors and submitted to polarizing cytokines, with and without spermidine treatment, to evaluate CD4 1 T-cell differentiation in vitro. Moreover, mice were subjected to oral supplementation of spermidine, or its

SummaryBrucella abortus elicits a vigorous Th1 immune response which activates cytotoxic T lymphocytes. However, B. abortus persists in its hosts in the presence of CD8 + T cells, establishing a chronic infection. Here, we report that B. abortus infection of human monocytes/macrophages inhibited the IFN-g-induced MHC-I cell surface expression. This phenomenon was dependent on metabolically active viable bacteria. MHC-I down-modulation correlated with the development of diminished CD8 + cytotoxic T cell response as evidenced by the reduced expression of the activation marker CD107a on CD8 + T lymphocytes and a diminished percentage of IFN-g-producing CD8+ T cells. Inhibition of MHC-I expression was not due to changes in protein synthesis. Rather, we observed that upon B. abortus infection MHC-I molecules were retained within the Golgi apparatus. Overall, these results describe a novel mechanism based on the intracellular sequestration of MHC-I molecules whereby B. abortus would avoid CD8 + cytotoxic T cell responses, evading their immunological surveillance.

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.

BackgroundCentral nervous system (CNS) invasion by bacteria of the genus Brucella results in an inflammatory disorder called neurobrucellosis. We have recently demonstrated that B. abortus infects microglia and astrocytes, eliciting the production of a variety of pro-inflammatory cytokines which contribute to CNS damage. Matrix metalloproteinases (MMP) have been implicated in inflammatory tissue destruction in a range of pathological situations in the CNS. Increased MMP secretion is induced by pro-inflammatory cytokines in a variety of CNS diseases characterized by tissue-destructive pathology.MethodsIn this study, the molecular mechanisms that regulate MMP secretion from Brucella-infected astrocytes in vitro were investigated. MMP-9 was evaluated in culture supernatants by ELISA, zymography and gelatinolytic activity. Involvement of mitogen-activated protein kinases (MAPK) signaling pathways was evaluated by Western blot and using specific inhibitors. The role of TNF-α was evaluated by ELISA and by assays with neutralizing antibodies.ResultsB. abortus infection induced the secretion of MMP-9 from murine astrocytes in a dose-dependent fashion. The phenomenon was independent of bacterial viability and was recapitulated by L-Omp19, a B. abortus lipoprotein model, but not its LPS. B. abortus and L-Omp19 readily activated p38 and Erk1/2 MAPK, thus enlisting these pathways among the kinase pathways that the bacteria may address as they invade astrocytes. Inhibition of p38 or Erk1/2 significantly diminished MMP-9 secretion, and totally abrogated production of this MMP when both MAPK pathways were inhibited simultaneously. A concomitant abrogation of B. abortus- and L-Omp19-induced TNF-α production was observed when p38 and Erk1/2 pathways were inhibited, indicating that TNF-α could be implicated in MMP-9 secretion. MMP-9 secretion induced by B. abortus or L-Omp19 was completely abrogated when experiments were conducted in the presence of a TNF-α neutralizing antibody. MMP-9 activity was detected in cerebrospinal fluid (CSF) samples from patients suffering from neurobrucellosis.ConclusionsOur results indicate that the inflammatory response elicited by B. abortus in astrocytes would lead to the production of MMP-9 and that MAPK may play a role in this phenomenon. MAPK inhibition may thus be considered as a strategy to control inflammation and CNS damage in neurobrucellosis.

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-1β, 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.

is an intracellular pathogen capable of surviving inside of macrophages. The success of 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 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 is able to down-regulate the expression of MHC-II, remained to be elucidated. In this study, we demonstrated that 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. 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 lipoprotein, diminished the expression of MHC-II and CIITA transcripts to the same extent as infection. IL-6 contributes to these down-regulatory phenomena. In addition, 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, 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 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.

bHuman brucellosis is a protean disease with a diversity of clinical signs and symptoms resulting from infection with Brucella species. Recent reports suggest a cross-regulation between adrenal steroids (cortisol and dehydroepiandrosterone [DHEA]) and the immune system. Monocytes and macrophages are the main replication niche for Brucella. Therefore, we investigated the role of adrenal hormones on the modulation of the immune response mediated by macrophages in B. abortus infection. Cortisol treatment during B. abortus infection significantly inhibits cytokine, chemokine, and MMP-9 secretion. In contrast, DHEA treatment had no effect. However, DHEA treatment increases the expression of costimulatory molecules (CD40, CD86), the adhesion molecule CD54, and major histocompatibility complex class I (MHC- Human brucellosis is characterized by a great diversity of clinical manifestations resulting from infection with Brucella species (4). It is chiefly an inflammatory disease. Inflammation is present both in the acute and chronic phases of the disease and in practically all of the organs affected. Clinical signs of such inflammation are undulant fever, endocarditis, arthritis, osteomyelitis, meningitis, pleocytosis, cellular infiltration of the joints, orchitis, nephritis, hepatic granuloma, etc. (5).IBrucella abortus infection elicits a vigorous Th1 immune response, which also activates cytotoxic T lymphocytes (6-9). Despite the recognition that Th1 immunity is crucial for the control of Brucella infection, several aspects of the immune response, i.e., how the innate immune response shapes the adaptive Th1 immune response and why Th1 immunity is not sufficient to completely eradicate the infection, constitute a conundrum. Macrophages/monocytes were recognized to play an important role in resistance to facultative intracellular bacteria such as Brucella, and activation of macrophages by specifically committed T lymphocytes formed the basis for cell-mediated immunity. However, B. abortus is endowed with a panoply of survival stratagems to evade immune responses and survive inside macrophages, creating a safe niche of replication inside the host (10-13).Several cytokines that are produced during B. abortus infec- [18][19][20]. In previous studies, significantly higher levels of cortisol in serum were reported in patients with acute brucellosis than in brucellosis patients at the end of antibiotic treatment or than in healthy subjects (21). In addition, adrenal glands are enlarged in patients with acute brucellosis but return to normal size after therapy (21). Of note, Th1 Citation Gentilini MV, Velásquez LN, Barrionuevo P, Arriola Benitez PC, Giambartolomei GH, Delpino MV. 2015. Adrenal steroids modulate the immune response during Brucella abortus infection by a mechanism that depends on the regulation of cytokine production.

Tuberculosis remains a major global health problem and efforts to develop a more effective vaccine have been unsuccessful so far. Targeting antigens (Ags) to dendritic cells (DCs) in vivo has emerged as a new promising vaccine strategy. In this approach, Ags are delivered directly to DCs via antibodies that bind to endocytic cell-surface receptors. Here, we explored DC-specific-ICAM3-grabbing-nonintegrin (DC-SIGN) targeting as a potential vaccine against tuberculosis. For this, we made use of the hSIGN mouse model that expresses human DC-SIGN under the control of the murine CD11c promoter. We show that in vitro and in vivo delivery of anti-DC-SIGN antibodies conjugated to Ag85B and peptide 25 of Ag85B in combination with anti-CD40, the fungal cell wall component zymosan, and the cholera toxin-derived fusion protein CTA1-DD induces strong Ag-specific CD4+ T-cell responses. Improved anti-mycobacterial immunity was accompanied by increased frequencies of Ag-specific IFN-γ+ IL-2+ TNF-α+ polyfunctional CD4+ T cells in vaccinated mice compared with controls. Taken together, in this study we provide the proof of concept that the human DC-SIGN receptor can be efficiently exploited for vaccine purposes to promote immunity against mycobacterial infections.