M. Victoria Delpino

Diminished Production of T Helper 1 Cytokines Correlates with T Cell Unresponsiveness to Brucella Cytoplasmic Proteins in Chronic Human Brucellosis

This study evaluated the cellular immune response against Brucella species cytoplasmic protein (CP) in peripheral blood mononuclear cells (PBMC) of 25 patients with brucellosis. In vitro proliferation and cytokine gene expression and production were investigated. PBMC from 14 patients proliferated in response to CP (responder patients [RPs]) and cells from 11 patients did not (nonresponder patients [NRPs]). CP-specific interleukin (IL)-2 and interferon-gamma were significantly induced in PBMC from RPs, compared with cells from NRPs. No significant differences were found in the production of IL-10 between the 2 groups. CP did not induce IL-4 production. A close relationship was observed between the clinical status of the patients and the T cell response against CP. Patient with acute infections responded to CP and induced production of T helper 1 (Th1) cytokines, whereas chronically infected patients did not. Diminished production of Th1 cytokines may contribute to T cell unresponsiveness in chronic human brucellosis.

Brucella abortus Inhibits Major Histocompatibility Complex Class II Expression and Antigen Processing through Interleukin-6 Secretion via Toll-Like Receptor 2

ABSTRACT The strategies that allow Brucella abortus to survive inside macrophages for prolonged periods and to avoid the immunological surveillance of major histocompatibility complex class II (MHC-II)-restricted gamma interferon (IFN-γ)-producing CD4+ T lymphocytes are poorly understood. We report here that infection of THP-1 cells with B. abortus inhibited expression of MHC-II molecules and antigen (Ag) processing. Heat-killed B. abortus (HKBA) also induced both these phenomena, indicating the independence of bacterial viability and involvement of a structural component of the bacterium. Accordingly, outer membrane protein 19 (Omp19), a prototypical B. abortus lipoprotein, inhibited both MHC-II expression and Ag processing to the same extent as HKBA. Moreover, a synthetic lipohexapeptide that mimics the structure of the protein lipid moiety also inhibited MHC-II expression, indicating that any Brucella lipoprotein could down-modulate MHC-II expression and Ag processing. Inhibition of MHC-II expression and Ag processing by either HKBA or lipidated Omp19 (L-Omp19) depended on Toll-like receptor 2 and was mediated by interleukin-6. HKBA or L-Omp19 also inhibited MHC-II expression and Ag processing of human monocytes. In addition, exposure to the synthetic lipohexapeptide inhibited Ag-specific T-cell proliferation and IFN-γ production of peripheral blood mononuclear cells from Brucella-infected patients. Together, these results indicate that there is a mechanism by which B. abortus may prevent recognition by T cells to evade host immunity and establish a chronic infection.

A bile salt hydrolase of Brucella abortus contributes to the establishment of a successful infection through the oral route in mice.

ABSTRACT Choloylglycine hydrolase (CGH), a bile salt hydrolase, has been annotated in all the available genomes of Brucella species. We obtained the Brucella CGH in recombinant form and demonstrated in vitro its capacity to cleave glycocholate into glycine and cholate. Brucella abortus 2308 (wild type) and its isogenic Δcgh deletion mutant exhibited similar growth rates in tryptic soy broth in the absence of bile. In contrast, the growth of the Δcgh mutant was notably impaired by both 5% and 10% bile. The bile resistance of the complemented mutant was similar to that of the wild-type strain. In mice infected through the intragastric or the intraperitoneal route, splenic infection was significantly lower at 10 and 20 days postinfection in animals infected with the Δcgh mutant than in those infected with the wild-type strain. For both routes, no differences in spleen CFU were found between animals infected with the wild-type strain and those infected with the complemented mutant. Mice immunized intragastrically with recombinant CGH mixed with cholera toxin (CGH+CT) developed a specific mucosal humoral (immunoglobulin G [IgG] and IgA) and cellular (interleukin-2) immune responses. Fifteen days after challenge by the same route with live B. abortus 2308 cells, splenic CFU counts were 10-fold lower in mice immunized with CGH+CT than in mice immunized with CT or phosphate-buffered saline. This study shows that CGH confers on Brucella the ability to resist the antimicrobial action of bile salts. The results also suggest that CGH may contribute to the ability of Brucella to infect the host through the oral route.

Brucella lipoproteins mimic dendritic cell maturation induced by Brucella abortus

Infection with Brucella abortus induces a pro-inflammatory response that drives T cell responses toward a Th1 profile. The mechanism by which this bacterium triggers this response is unknown. Dendritic cells (DC) are crucial mediators at the host-pathogen interface and are potent Th1-inducing antigen-presenting cells. Thus, we examined the mechanism whereby B. abortus stimulate human DC maturation. B. abortus-infected DC increased the expression of CD86, CD80, CCR7, CD83, MHCII, MHCI and CD40 and induced the production of TNF-alpha, IL-6, IL-10 and IL-12. Both phenomena were not dependent on bacterial viability since they were also induced by heat-killed B. abortus (HKBA). B. abortus LPS was unable to induce markers up-regulation or cytokine production. We next investigated the capacity of the outer membrane protein 19 (Omp19) as a B. abortus lipoprotein model to induce DC maturation. Lipidated Omp19 (L-Omp19), but not its unlipidated form, increased the expression of cell surface markers and the secretion of cytokines. L-Omp19-matured DC also have decreased endocytic activity and displayed enhanced T cell stimulatory activity in a MLR. Pre-incubation of DC with anti-TLR2 mAb blocked L-Omp19-mediated cytokine production. These results demonstrate that B. abortus lipoproteins can stimulate DC maturation providing a mechanism by which these bacteria generate a Th1-type immune response.

Brucella abortus Induces the Secretion of Proinflammatory Mediators from Glial Cells Leading to Astrocyte Apoptosis

Central nervous system (CNS) invasion by bacteria of the genus Brucella results in an inflammatory disorder called neurobrucellosis. In this study we present in vivo and in vitro evidence that B. abortus and its lipoproteins activate the innate immunity of the CNS, eliciting an inflammatory response that leads to astrogliosis, a characteristic feature of neurobrucellosis. Intracranial injection of heat-killed B. abortus (HKBA) or outer membrane protein 19 (Omp19), a B. abortus lipoprotein model, induced astrogliosis in mouse striatum. Moreover, infection of astrocytes and microglia with B. abortus induced the secretion of interleukin (IL)-6, IL-1beta, tumor necrosis factor (TNF)-alpha, macrophage chemoattractant protein-1, and KC (CXCL1). HKBA also induced these inflammatory mediators, suggesting the involvement of a structural component of the bacterium. Accordingly, Omp19 induced the same cytokine and chemokine secretion pattern. B. abortus infection induced astrocyte, but not microglia, apoptosis. Indeed, HKBA and Omp19 elicited not only astrocyte apoptosis but also proliferation, two features observed during astrogliosis. Apoptosis induced by HKBA and L-Omp19 was completely suppressed in cells of TNF receptor p55-/- mice or when the general caspase inhibitor Z-VAD-FMK was added to cultures. Hence, TNF-alpha signaling via TNF receptor (TNFR) 1 through the coupling of caspases determines apoptosis. Our results provide proof of the principle that Brucella lipoproteins could be key virulence factors in neurobrucellosis and that astrogliosis might contribute to neurobrucellosis pathogenesis.

Macrophage-elicited osteoclastogenesis in response to Brucella abortus infection requires TLR2/MyD88-dependent TNF-α production

Osteoarticular complications are common in human brucellosis, but the pathogenic mechanisms involved are largely unknown. In this manuscript, we described an immune mechanism for inflammatory bone loss in response to infection by Brucella abortus. We established a requirement for MyD88 and TLR2 in TNF‐α‐elicited osteoclastogenesis in response to B. abortus infection. CS from macrophages infected with B. abortus induced BMM to undergo osteoclastogenesis. Although B. abortus‐infected macrophages actively secreted IL‐1β, IL‐6, and TNF‐α, osteoclastogenesis depended on TNF‐α, as CS from B. abortus‐infected macrophages failed to induce osteoclastogenesis in BMM from TNFRp55–/– mice. CS from B. abortus‐stimulated MyD88–/– and TLR2–/– macrophages failed to express TNF‐α, and these CS induced no osteoclast formation compared with that of the WT or TLR4–/– macrophages. Omp19, a B. abortus lipoprotein model, recapitulated the cytokine production and subsequent osteoclastogenesis induced by the whole bacterium. All phenomena were corroborated using human monocytes, indicating that this mechanism could play a role in human osteoarticular brucellosis. Our results indicate that B. abortus, through its lipoproteins, may be involved in bone resorption through the pathological induction of osteoclastogenesis.

Potential Role of Fibroblast-Like Synoviocytes in Joint Damage Induced by Brucella abortus Infection through Production and Induction of Matrix Metalloproteinases

ABSTRACT Arthritis is one of the most common complications of human brucellosis, but its pathogenic mechanisms have not been elucidated. Fibroblast-like synoviocytes (FLS) are known to be central mediators of joint damage in inflammatory arthritides through the production of matrix metalloproteinases (MMPs) that degrade collagen and of cytokines and chemokines that mediate the recruitment and activation of leukocytes. In this study we show that Brucella abortus infects and replicates in human FLS (SW982 cell line) in vitro and that infection results in the production of MMP-2 and proinflammatory mediators (interleukin-6 [IL-6], IL-8, monocyte chemotactic protein 1 [MCP-1], and granulocyte-macrophage colony-stimulating factor [GM-CSF]). Culture supernatants from Brucella-infected FLS induced the migration of monocytes and neutrophils in vitro and also induced these cells to secrete MMP-9 in a GM-CSF- and IL-6-dependent fashion, respectively. Reciprocally, culture supernatants from Brucella-infected monocytes and neutrophils induced FLS to produce MMP-2 in a tumor necrosis factor alpha (TNF-α)-dependent fashion. The secretion of proinflammatory mediators and MMP-2 by FLS did not depend on bacterial viability, since it was also induced by heat-killed B. abortus (HKBA) and by a model Brucella lipoprotein (L-Omp19). These responses were mediated by the recognition of B. abortus antigens through Toll-like receptor 2. The intra-articular injection of HKBA or L-Omp19 into the knee joint of mice resulted in the local induction of the proinflammatory mediators MMP-2 and MMP-9 and in the generation of a mixed inflammatory infiltrate. These results suggest that FLS, and phagocytes recruited by them to the infection focus, may be involved in joint damage during brucellar arthritis through the production of MMPs and proinflammatory mediators.

The TolC Homologue of Brucella suis Is Involved in Resistance to Antimicrobial Compounds and Virulence

ABSTRACT Brucella spp., like other pathogens, must cope with the environment of diverse host niches during the infection process. In doing this, pathogens evolved different type of transport systems to help them survive and disseminate within the host. Members of the TolC family have been shown to be involved in the export of chemically diverse molecules ranging from large protein toxins to small toxic compounds. The role of proteins from the TolC family in Brucella and otherα -2-proteobacteria has been explored little. The gene encoding the unique member of the TolC family from Brucella suis (BepC) was cloned and expressed in an Escherichia coli mutant disrupted in the gene encoding TolC, which has the peculiarity of being involved in diverse transport functions. BepC fully complemented the resistance to drugs such as chloramphenicol and acriflavine but was incapable of restoring hemolysin secretion in the tolC mutant of E. coli. An insertional mutation in the bepC gene strongly affected the resistance phenotype of B. suis to bile salts and toxic chemicals such as ethidium bromide and rhodamine and significantly decreased the resistance to antibiotics such as erythromycin, ampicillin, tetracycline, and norfloxacin. Moreover, the B. suis bepC mutant was attenuated in the mouse model of infection. Taken together, these results suggest that BepC-dependent efflux processes of toxic compounds contribute to B. suis survival inside the host.

Brucella-infected hepatocytes mediate potentially tissue-damaging immune responses

BACKGROUND & AIMS Hepatic involvement is frequent in human brucellosis. While different histopathological lesions have been reported in these patients, the underlying cellular and molecular mechanisms have not been addressed. METHODS This study assessed whether Brucella abortus can infect a human hepatoma cell line and induce a proinflammatory response in these cells. RESULTS The bacterium not only infected the human hepatoma cell line HepG2 but also exhibited intracellular replication. The infection induced hepatoma cells to secrete IL-8, and supernatants from Brucella-infected hepatoma cells were shown to induce the migration of human neutrophils. The infection also induced the expression of the intercellular adhesion molecule ICAM-1 on hepatoma cells, and the adhesion of neutrophils to these cells was significantly higher than to uninfected hepatoma cells. ICAM-1 expression was also induced by stimulation of hepatoma cells with supernatants from Brucella-infected neutrophils. While Brucella infection did not induce the expression of matrix metalloproteinases (MMPs) in hepatoma cells, it significantly induced MMP-9 in neutrophils. Hepatoma cell apoptosis was significantly induced by B. abortus infection and also by stimulation with supernatants from Brucella-infected neutrophils. CONCLUSIONS The present study provides clues regarding potential mechanisms of tissue damage during liver brucellosis.

Occupational infection due to Brucella abortus S19 among workers involved in vaccine production in Argentina

The pathological consequences of exposure to the vaccine strain Brucella abortus S19 were evaluated in 30 employees from vaccine-manufacturing plants. Active brucellosis was diagnosed in 21 subjects, of whom only five recalled an accidental exposure. Clinical manifestations were mild, and only one patient presented a complication. After antimicrobial therapy, initially symptomatic patients either experienced clinical remission or had mild persistent symptoms. This is the first study reporting infection by B. abortus S19 among workers from vaccine-manufacturing plants, which in many cases was acquired from unnoticed exposures. Measures to improve the safety of B. abortus S19 handling should be implemented.