Immunoproteasome Subunits Are Required for CD8+ T Cell Function and Host Resistance to Brucella abortus Infection in Mice.

The immunoproteasome is a specific proteasome isoform composed of three subunits, termed ß1i, ß2i, and ß5i. Its proteolytic activity enhances the quantity and quality of peptides to be presented by major histocompatibility complex class I (MHC-I) molecules to CD8+ T cells. However, the role of the combined deficiency of the three immunoproteasome subunits in protective immunity against bacterial pathogens has not been investigated. In this study, we addressed the role of the immunoproteasome during infection by Brucella abortus, an intracellular bacterium that requires CD8+ T cell responses for the control of infection. Here, we demonstrate that immunoproteasome triple-knockout (TKO) mice were more susceptible to Brucella infection. This observed susceptibility was accompanied by reduced interferon gamma (IFN-γ) production by mouse CD4+ and CD8+ T lymphocytes. Moreover, the absence of the immunoproteasome had an impact on MHC-I surface expression and antigen presentation by dendritic cells. CD8+ T cell function, which plays a pivotal role in B. abortus immunity, also presented a partial impairment of granzyme B expression and, consequently, reduced cytotoxic activity. In conclusion, these results strongly suggest that immunoproteasome subunits are important components in host resistance to B. abortus infection by impacting both the magnitude and quality of CD8+ T cell responses.

Pyruvate kinase is necessary for Brucella abortus full virulence in BALB/c mouse.

Brucellosis, caused by a facultative intracellular pathogen Brucella, is one of the most prevalent zoonosis worldwide. Host infection relies on several uncanonical virulence factors. A recent research hotpot is the links between carbon metabolism and bacterial virulence. In this study, we found that a carbon metabolism-related pyruvate kinase (Pyk) encoded by pyk gene (locus tag BAB_RS24320) was associated with Brucella virulence. Determination of bacterial growth curves and resistance to environmental stress factors showed that Pyk plays an important role in B. abortus growth, especially under the conditions of nutrition deprivation, and resistance to oxidative stress. Additionally, cell infection assay showed that Pyk is necessary for B. abortus survival and evading fusion with lysosomes within RAW264.7 cells. Moreover, animal experiments exhibited that the Pyk deletion significantly reduced B. abortus virulence in a mouse infection model. Our results elucidated the role of the Pyk in B. abortus virulence and provided information for further investigation of Brucella virulence associated carbon metabolism.

Increased Plasma Levels of Heme Oxygenase-1 in Human Brucellosis.

BACKGROUND: Brucellosis is associated with inflammation and the oxidative stress response. Heme oxygenase-1 (HO-1) is a cytoprotective stress-responsive enzyme that has anti-inflammatory and anti-oxidant effects. Nevertheless, the role of HO-1 in human brucellosis has not yet been studied. The aim of this study was to examine the plasma levels of HO-1 in patients with brucellosis and to evaluate the ability of plasma HO-1 levels as an auxiliary diagnosis, a severity predictor, and a monitor for brucellosis treatments. METHODS: A total of 75 patients with brucellosis were divided into the acute, subacute, chronic active, and chronic stable groups. An additional 20 volunteers were included as the healthy control group. The plasma HO-1 levels and other laboratory parameters were measured in all groups. Furthermore, the plasma levels of HO-1 in the acute group were compared before and after treatment. RESULTS: The plasma HO-1 levels were considerably increased in the acute (4.97 ± 3.55), subacute (4.98 ± 3.23), and chronic active groups (4.43 ± 3.00) with brucellosis compared to the healthy control group (1.03 ± 0.63) (p < 0.01). In the acute group, the plasma HO-1 levels in the post-treatment group (2.33 ± 2.39) were significantly reduced compared to the pre-treatment group (4.97 ± 3.55) (p < 0.01). On the other hand, the plasma HO-1 levels were higher in the chronic active group (4.43 ± 3.00) than the chronic stable group (2.74 ± 2.23) (p < 0.05). However, the plasma HO-1 levels in the chronic stable group (2.74 ± 2.23) remained higher than the levels in the healthy control group (1.03 ± 0.63) (p < 0.05). The HO-1 levels were positively correlated with the C-reactive protein (CRP) levels in patients with brucellosis (r = 0.707, p < 0.01). CONCLUSIONS: The plasma HO-1 levels can reflect patients' brucellosis status and may be used as a supplementary plasma marker for diagnosing brucellosis and monitoring its treatment.

5-Lipoxygenase negatively regulates Th1 response during Brucella abortus infection in mice.

Brucella abortus is a Gram-negative bacterium that infects humans and cattle, causing a chronic inflammatory disease known as brucellosis. A Th1-mediated immune response plays a critical role in host control of this pathogen. Recent findings indicate contrasting roles for lipid mediators in host responses against infections. 5-Lipoxygenase (5-LO) is an enzyme required for the production of the lipid mediators leukotrienes and lipoxins. To determine the involvement of 5-LO in host responses to B. abortus infection, we intraperitoneally infected wild-type and 5-LO-deficient mice and evaluated the progression of infection and concomitant expression of immune mediators. Here, we demonstrate that B. abortus induced the upregulation of 5-LO mRNA in wild-type mice. Moreover, this pathogen upregulated the production of the lipid mediators leukotriene B4 and lipoxin A4 in a 5-LO-dependent manner. 5-LO-deficient mice displayed lower bacterial burdens in the spleen and liver and less severe liver pathology, demonstrating an enhanced resistance to infection. Host resistance paralleled an increased expression of the proinflammatory mediators interleukin-12 (IL-12), gamma interferon (IFN-γ), and inducible nitric oxide synthase (iNOS) during the course of infection. Moreover, we demonstrated that 5-LO downregulated the expression of IL-12 in macrophages during B. abortus infection. Our results suggest that 5-LO has a major involvement in B. abortus infection, by functioning as a negative regulator of the protective Th1 immune responses against this pathogen.

Potential role of fibroblast-like synoviocytes in joint damage induced by Brucella abortus infection through production and induction of matrix metalloproteinases.

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.

Comparative analysis of cerebrospinal fluid adenosine deaminase activity in meningitis.

AIM: The purpose is to determine the cut-off value of adenosine deaminase (ADA) activity in cerebrospinal fluid (CSF) of patients with tuberculous and non-tuberculous meningitis, and to assess its value in differential diagnosis. MATERIAL AND METHODS: This study was conducted in 91 patients with meningitis in two university hospitals in Turkey. 24 patients had tuberculous meningitis (TBM), 25 purulent meningitis (PM), 25 aseptic meningitis (AM) and 17 neurobrucellosis (BM). ADA activity of CSF was quantified by colorimetry. RESULTS: In our study, mean ADA values in CSF were 28.34 ± 14.83 IU/L in TB cases, 8.71 ± 5.83 IU/L in BM, 6.18 ± 2.54 IU/L in PM and 3.43 ± 3.48 U/L in AM cases. If we accept for CSF ADA an activity cut-off value of 12.5 IU/L for differential diagnosis of TBM and BM, its sensitivity was 92% and specificity was 88%. If we accept 12.35 IU/L for differential diagnosis of TBM and PM, its sensitivity was 92% and specificity was 100%. If we accept 6.45 IU/L for differential diagnosis of TBM and AM, its sensitivity was 100% and specificity was 92%. Additionally, we examined the cases after dividing them into two groups, viz. TB and non-TB. If we accept an ADA activity cut-off level of 11 IU/L for differential diagnosis of TB and non-TB by applying ROC analysis, its sensitivity was 92% and specificity was 90%. CONCLUSION: The sensitivity and specificity for CSF ADA activity are markedly high in differential diagnosis of TB from non-TB. Hence CSF ADA activity may be used as a simple, cost-effective and reliable test for early differential diagnosis of TB.

Evaluating the validity of serum neopterin and chitotriosidase levels in follow-up brucellosis patients.

OBJECTIVE: Due to its high morbidity rates brucellosis, a systemic inflammatory disease, is still an important health problem, particularly in Mediterranean regions. One-third of the patients are characterized with musculoskeletal involvement. Principally in chronic cases, there are difficulties in the follow-up of therapy success. Radiological imaging methods are used in musculoskeletal brucellosis in addition to standard serological tests. Two macrophage products, namely neopterin (NPT) and chitotriosidase (ChT), are used as novel markers in order to reflect the status of inflammatory diseases. In this study, we aimed to test the validity of these markers in follow-up of patients with brucellosis. PATIENTS AND METHODS: A total of 40 brucellosis cases were included in the study and 27 healthy individuals were used as controls. Twenty of the brucellosis patients were presented with sacroiliac joint involvement. A 6-week treatment of doxycycline combined with rifampicin or streptomycin was used to treat brucellosis. Clinical observations and serological outcome were used to determine whether treatment was successful or not. RESULTS: All of the 20 brucellosis patients without musculoskeletal involvement healed with the first cure of treatment, but all of the brucella-sacroiliitis patients had to be retreated. In addition to routine testing, serum NPT and ChT levels were evaluated after each treatment. The results presented a clear fall in both NPT and ChT levels in parallel with the serological data of the patients. CONCLUSION: In conclusion, NPT as well as ChT seems to be a useful marker in the follow-up of brucellosis patients and for evaluating the success of therapy.

Mice lacking components of adaptive immunity show increased Brucella abortus virB mutant colonization.

The Brucella abortus type IV secretion system (T4SS), encoded by the virB genes, is essential for survival in mononuclear phagocytes in vitro. In the mouse model, a B. abortus virB mutant was initially able to colonize the spleen at the level of the wild type for approximately 3 to 5 days, which coincided with the development of adaptive immunity. To investigate the relationship between survival in macrophages cultivated in vitro and persistence in tissues in vivo, we tested the ability of mutant mice lacking components of adaptive immunity to eliminate the virB mutant from the spleen during a mixed infection with the B. abortus wild type. Ifng(-/-) or beta(2)m(-/-) mice were able to clear the virB mutant to the same degree as control mice. However, spleens of Rag1(-/-) mice and Igh6(-/-) mice were more highly colonized by the virB mutant than control mice after 14 to 21 days, suggesting that, in these mice, there is not an absolute requirement for the T4SS to mediate persistence of B. abortus in the spleen. Macrophages isolated from Igh6(-/-) mice killed the virB mutant to the same extent as macrophages from control mice, showing that the reduced ability of these mice to clear the virB mutant from the spleen does not correlate with diminished macrophage function in vitro. These results show that in the murine model host, the T4SS is required for persistence beyond 3 to 5 days after infection and suggest that the T4SS may contribute to evasion of adaptive immune mechanisms by B. abortus.

Different roles of the two high-oxygen-affinity terminal oxidases of Brucella suis: Cytochrome c oxidase, but not ubiquinol oxidase, is required for persistence in mice.

The survival of Brucella suis mutant strains in mice demonstrated different roles of the two high-oxygen-affinity terminal oxidases. The cbb3-type cytochrome c oxidase was essential for chronic infection in oxygen-deficient organs. Lack of the cytochrome bd ubiquinol oxidase led to hypervirulence of bacteria, which could rely on nitrite accumulation inhibiting the inducible nitric oxide synthase of the host.

Evaluation of Brucella abortus DNA vaccine by expression of Cu-Zn superoxide dismutase antigen fused to IL-2.

The Cu-Zn superoxide dismutase (SOD) antigen of Brucella abortus was previously identified to be a T cell antigen which induces both proliferation of and gamma interferon (IFN-gamma) secretion by T cells from infected mice. In an earlier study, we demonstrated that intramuscular injection of mice with a plasmid DNA carrying the gene for SOD leads to the development of significant protection against B. abortus challenge. It has been reported that the antigen-specific immune responses generated by a DNA vaccine can be enhanced by co-delivery of certain cytokine genes. In this study, we evaluated the effect of delivering IL-2 on the efficacy of SOD DNA vaccine by generating a plasmid (pSecTag-SOD-IL2) that codes for a secretory fusion protein of SOD and IL-2. Another plasmid (pSecTag-SOD) that codes for only SOD as a secretory protein was used for comparison. BALB/c mice injected intramuscularly with pSecTag-SOD or pSecTag-SOD-IL2, but not the control plasmid pSecTag, developed SOD-specific antibody and T cell immune responses. Upon in vitro stimulation with recombinant SOD (rSOD) antigen, T cells from mice immunized with pSecTag-SOD-IL2, in comparison with those from mice immunized with pSecTag-SOD, exhibited a lower proliferation response but produced significantly higher concentrations of IFN-gamma. Both DNA vaccines, however, induced similar levels of SOD-specific antibodies and cytotoxic T cell response. Although mice immunized with pSecTag-SOD-IL2 showed increased resistance to challenge with B. abortus virulent strain 2308, this increase was not statistically significant from that of pSecTag-SOD vaccinated mice. These results suggest that a SOD DNA vaccine fused to IL2 did not improve protection efficacy.

Increased pleural fluid adenosine deaminase in brucellosis is difficult to differentiate from tuberculosis.

Pleural involvement in brucellosis is very rare. Current knowledge on brucella pleuritis is limited to a few case studies, and pleural adenosine deaminase (ADA) in brucellosis has not been studied previously. We report the pleural fluid characteristics, including ADA, of two cases with brucella pleurisy. Analysis of the pleural fluids revealed exudative effusions with increased ADA level, decreased glucose concentration, and lymphocyte predominance. The similarity with tuberculous pleurisy was remarkable. We suggest that brucellosis should be considered in the differential diagnosis of tuberculosis, especially in regions endemic for both diseases.

virB-Mediated survival of Brucella abortus in mice and macrophages is independent of a functional inducible nitric oxide synthase or NADPH oxidase in macrophages.

The Brucella abortus virB locus is required for establishing chronic infection in the mouse. Using in vitro and in vivo models, we investigated whether virB is involved in evasion of the bactericidal activity of NADPH oxidase and the inducible nitric oxide synthase (iNOS) in macrophages. Elimination of NADPH oxidase or iNOS activity in macrophages in vitro increased recovery of wild-type B. abortus but not recovery of a virB mutant. In mice lacking either NADPH oxidase or iNOS, however, B. abortus infected and persisted to the same extent as it did in congenic C57BL/6 mice up until 60 days postinfection, suggesting that these host defense mechanisms are not critical for limiting bacterial growth in the mouse. A virB mutant did not exhibit increased survival in either of the knockout mouse strains, indicating that this locus does not contribute to evasion of nitrosative or oxidative killing mechanisms in vivo.

Identification of the perosamine synthetase gene of Brucella melitensis 16M and involvement of lipopolysaccharide O side chain in Brucella survival in mice and in macrophages.

Brucella organisms are facultative intracellular bacteria that may infect many species of animals as well as humans. The smooth lipopolysaccharide (S-LPS) has been reported to be an important virulence factor of these organisms, but the genetic basis of expression of the S-LPS O antigen has not yet been described. Likewise, the role of the O side chain of S-LPS in the survival of Brucella has not been clearly defined. A mini-Tn5 transposon mutant library of Brucella melitensis 16M was screened by enzyme-linked immunosorbent assay (ELISA) with monoclonal antibodies (MAbs) directed against the O side chain of Brucella. One mutant, designated B3B2, failed to express any O side chain as confirmed by ELISA, Western blot analysis, and colony coloration with crystal violet. Nucleotide sequence analysis demonstrated that the transposon disrupted an open reading frame with significant homology to the putative perosamine synthetase genes of Vibrio cholerae O1 and Escherichia coli O157:H7. The low G+C content of this DNA region suggests that this gene may have originated from a species other than a Brucella sp. The survival of B. melitensis mutant strain B3B2 in the mouse model and in bovine macrophages was examined. The results suggested that S-LPS or, more precisely, its O side chain is essential for survival in mice but not in macrophages.

[Activity of adenosine deaminase in acute brucellosis and complicated brucellosis]. / Estudio de la actividad de adenosina desaminasa en la brucelosis aguda y en la brucelosis complicada.

BACKGROUND: Adenosine deaminase (ADA) is an essential enzyme for the differentiation and proliferation of T lymphocytes and the monocyte-macrophage system. The basic immunitary response of brucellosis is cellular. To this end, ADA activity was evaluated in brucellosis. METHODS: Serum ADA activity was assessed by a colorimetric method in 67 patients with brucellosis, before therapy and at 1, 3 and 6 months of follow up. RESULTS: Serum ADA activity in brucellosis was higher than that in 52 healthy controls, both in those with the acute febrile noncomplicated form (48 patients) and in those with focal symptoms from one organ (19 patients) (p less than 0.0001 and p less than 0.001). There were no differences between both groups of brucellosis. There was a negative correlation between the duration of the disease and ADA activity. After therapy there was a rapid decrease of ADA values, more marked in patients with noncomplicated brucellosis. During the follow up, only one patient had a new increase in ADA activity, coincident with a clinical and bacteriological relapse, and previous to the increase of IgG anti-Brucella titers. CONCLUSIONS: The results indicate that adenosine deaminase activity is increased during the active stage of brucellosis. It can be considered as a biochemical follow up marker of the disease and, probably, as a marker of relapses.

[Comparative cytoenzymatic study of infectious and vaccinal processes in experimental brucellosis]. / Sravnitel'noe tsitoénzimaticheskoe issledovanie infektsionnogo i vaktsinnogo protsessov pri éksperimental'nom brutselleze.

In experiments on 240 guinea-pigs metabolic changes were found to occur in peripheral blood cells in the process of the development of brucellosis infection and after immunization. The degree and character of the activity of lysosomal enzymes depended on the time of infection and immunity formation. In comparison with the vaccinal culture of Br. abortus 19, the virulent culture of Br. abortus 544 induced greater changes in the activity of esterase, acidic and alkaline phosphatase in neutrophils, in the activity of acidic phosphatase in lymphocytes, as well as in the number of lymphocytes containing this enzyme. The data on enzymatic activity are recommended for use as a differential test for evaluating the character of the infectious and vaccinal processes.

Characterization of macrophage functions in mice infected with Brucella abortus.

Macrophage spreading, surface receptor density/avidity, phagocytosis, random migration, chemotactic responsiveness, and serum lysozyme were examined during the course of infection (up to 60 days) of mice with Brucella abortus strain 19. Markedly enhanced in vitro spreading activity was observed throughout the period of study. The density/avidity of cell surface immunoglobulin G Fc receptors was increased for up to 60 days postinfection. Internalization of sheep erythrocytes via C3 receptors was significantly enhanced. Random locomotion and chemotactic responsiveness to lymphocyte-derived chemotactic factor and N-formyl-L-methionyl-L-leucyl-L-phenylalanine were markedly stimulated. Serum lysozyme was also elevated in infected animals. These changes indicated significant and prolonged enhancement of macrophage activity during Brucella infection. These findings are discussed in relation to previous reports describing macrophage activation by Brucella.

[Lysozyme concentration in the loose connective tissue of animals with different species sensitivity to brucellosis]. / Soderzhanie lizotsima v rykhloi soedinitel'noi tkani u zhivotnykh s razlichnoi vidovoi chuvstvitel'nost'iu k brutselleznoi infektsii.

Lysozyme content was determined in the subcutaneous cellular tissue of guinea pigs and albino rats differing by species resistance to brucella infection. Experiments were conducted on intact animals and those inoculated subcutaneously with live Br. abortus 19-BA vaccine. Connective tissue was taken from the site of the vaccine administration and from the contralateral side (control). Observations showed connective tissue of the animals highly sensitive to brucella infection to be exceedingly poor in this enzyme; as to connective tissue of albino rats with low sensitivity--it contained high amount of this enzyme. Lysozyme content increased considerably in the animals belonging to both species in the inflammatory focus developing at the site of the vaccine inoculation.