Group G Streptococcus Induces an Autoimmune Carditis Mediated by Interleukin 17A and Interferon γ in the Lewis Rat Model of Rheumatic Heart Disease.

Acute rheumatic fever and rheumatic heart disease (ARF/RHD) have long been described as autoimmune sequelae of Streptococcus pyogenes or group A streptococcal (GAS) infection. Both antibody and T-cell responses against immunodominant GAS virulence factors, including M protein, cross-react with host tissue proteins, triggering an inflammatory response leading to permanent heart damage. However, in some ARF/RHD-endemic regions, throat carriage of GAS is low. Because Streptococcus dysgalactiae subspecies equisimilis organisms, also known as ß-hemolytic group C streptococci and group G streptococci (GGS), also express M protein, we postulated that streptococci other than GAS may have the potential to initiate or exacerbate ARF/RHD. Using a model initially developed to investigate the uniquely human disease of ARF/RHD, we have discovered that GGS causes interleukin 17A/interferon γ-induced myocarditis and valvulitis, hallmarks of ARF/RHD. Remarkably the histological, immunological, and functional changes in the hearts of rats exposed to GGS are identical to those exposed to GAS. Furthermore, antibody cross-reactivity to cardiac myosin was comparable in both GGS- and GAS-exposed animals, providing additional evidence that GGS can induce and/or exacerbate ARF/RHD.

Repeat exposure to group A streptococcal M protein exacerbates cardiac damage in a rat model of rheumatic heart disease.

Rheumatic fever and rheumatic heart disease (RF/RHD) develop following repeated infection with group A streptococci (GAS). We used the Rat Autoimmune Valvulitis (RAV) model of RF/RHD to demonstrate that repetitive booster immunization with GAS-derived recombinant M protein (rM5) resulted in an enhanced anti-cardiac myosin antibody response that may contribute to the breaking of immune tolerance leading to RF/RHD and increased infiltration of heart valves by mononuclear cells. With each boost, more inflammatory cells were observed infiltrating heart tissue which could lead to severe cardiac damage. We also found evidence that both complement and anti-M protein antibodies in serum from rM5-immunized rats have the potential to contribute to inflammation in heart valves by activating cardiac endothelium. More importantly, we have demonstrated by electrocardiography for the first time in the RAV model that elongation of P-R interval follows repetitive boost with rM5. Our observations provide experimental evidence for cardiac alterations following repeated exposure to GAS M protein with immunological and electrophysiological features resembling that seen in humans following recurrent GAS infection.

Plasmacytoid dendritic cell bactericidal activity against Burkholderia pseudomallei.

Melioidosis sepsis, caused by Burkholderia pseudomallei, is associated with high mortality due to an overwhelming inflammatory response. Plasmacytoid dendritic cells (pDC) are potent producers of type I interferons (IFN). This study investigated whether pDC and type I IFN play a role during the early stages of B. pseudomallei infection. Human and murine pDC internalised and killed B. pseudomallei as efficiently as murine conventional DC (cDC). pDC derived from B. pseudomallei-susceptible (BALB/c) mice demonstrated poor intracellular killing and increased IFN-alpha compared to pDC derived from B. pseudomallei-resistant (C57BL/6) mice. This is the first evidence of pDC bactericidal activity against B. pseudomallei infection.

Animal models to investigate the pathogenesis of rheumatic heart disease.

Rheumatic fever (RF) and rheumatic heart disease (RHD) are sequelae of group A streptococcal (GAS) infection. Although an autoimmune process has long been considered to be responsible for the initiation of RF/RHD, it is only in the last few decades that the mechanisms involved in the pathogenesis of the inflammatory condition have been unraveled partly due to experimentation on animal models. RF/RHD is a uniquely human condition and modeling this disease in animals is challenging. Antibody and T cell responses to recombinant GAS M protein (rM) and the subsequent interactions with cardiac tissue have been predominantly investigated using a rat autoimmune valvulitis model. In Lewis rats immunized with rM, the development of hallmark histological features akin to RF/RHD, both in the myocardial and in valvular tissue have been reported, with the generation of heart tissue cross-reactive antibodies and T cells. Recently, a Lewis rat model of Sydenham's chorea and related neuropsychiatric disorders has also been described. Rodent models are very useful for assessing disease mechanisms due to the availability of reagents to precisely determine sequential events following infection with GAS or post-challenge with specific proteins and or carbohydrate preparations from GAS. However, studies of cardiac function are more problematic in such models. In this review, a historical overview of animal models previously used and those that are currently available will be discussed in terms of their usefulness in modeling different aspects of the disease process. Ultimately, cardiologists, microbiologists, immunologists, and physiologists may have to resort to diverse models to investigate different aspects of RF/RHD.

Migration of dendritic cells facilitates systemic dissemination of Burkholderia pseudomallei.

Burkholderia pseudomallei, the etiological agent for melioidosis, is an important cause of community-acquired sepsis in northern Australia and northeast Thailand. Due to the rapid dissemination of disease in acute melioidosis, we hypothesized that dendritic cells (DC) could act as a vehicle for dissemination of B. pseudomallei. Therefore, this study investigated the effect of B. pseudomallei infection on DC migration capacity and whether migration of DC enabled transportation of B. pseudomallei from the site of infection. B. pseudomallei stimulated significantly increased migration of bone marrow-derived DC (BMDC), both in vitro and in vivo, compared to uninfected BMDC. Furthermore, migration of BMDC enabled significantly increased in vitro trafficking of B. pseudomallei and in vivo dissemination of B. pseudomallei to secondary lymphoid organs and lungs of C57BL/6 mice. DC within the footpad infection site of C57BL/6 mice also internalized B. pseudomallei and facilitated dissemination. Although DC have previously been shown to kill intracellular B. pseudomallei in vitro, the findings of this study demonstrate that B. pseudomallei-infected DC facilitate the systemic spread of this pathogen.

Improved diagnosis of melioidosis using a 2-dimensional immunoarray.

Melioidosis is caused by the Gram negative bacterium Burkholderia pseudomallei. The gold standard for diagnosis is culture, which requires at least 3-4 days obtaining a result, hindering successful treatment of acute disease. The existing indirect haemagglutination assay (IHA) has several disadvantages, in that approximately half of patients later confirmed culture positive are not diagnosed at presentation and a subset of patients are persistently seronegative. We have developed 2 serological assays, an enzyme-linked immunosorbent assay (ELISA), and a 2-dimensional immunoarray (2DIA), capable of detecting antibodies in patient sera from a greater proportion of IHA-negative patient subsets. The 2DIA format can distinguish between different LPS serotypes. Currently, the 2DIA has a sensitivity and specificity of 100% and 87.1%, respectively, with 100% of culture-positive, IHA-negative samples detected. The ELISA has a sensitivity and specificity of 86.2% and 93.5%, respectively, detecting 67% of culture-positive, IHA-negative samples. The ELISA and 2DIA tests described here are more rapid and reliable for serological testing compared to the existing IHA.

ELISA and immuno-polymerase chain reaction assays for the sensitive detection of melioidosis.

Melioidosis is caused by the Gram-negative bacterium Burkholderia pseudomallei. The gold standard for diagnosis is culture, which requires at least 3-4 days to obtain a result, hindering successful treatment of acute disease. An indirect haemagglutination assay (IHA) is often used but lacks sensitivity. Approximately half of patients later confirmed culture positive are not detected by IHA at presentation and a subset of patients persistently continue to be IHA negative. More rapid and reliable serologic testing for melioidosis is essential and will improve diagnosis and patient outcome. We have developed an ELISA and a quantitative immuno-polymerase chain reaction assay capable of detecting melioidosis-specific antibodies and demonstrate their validity with IHA-negative sera from patients with melioidosis. These new sensitive assays are based upon a secreted antigenic fraction from B. pseudomallei and will be ideal for the diagnosis of melioidosis in patients in nonendemic regions returning from endemic tropical areas and for seroepidemiologic surveys.

Evidence of Burkholderia pseudomallei-specific immunity in patient sera persistently nonreactive by the indirect hemagglutination assay.

The indirect hemagglutination assay (IHA) is the most frequently used serological test to confirm exposure to Burkholderia pseudomallei. Patients with culture-confirmed disease often have a nonreactive IHA at presentation and occasionally fail to seroconvert on serial testing. We investigated whether using antigens derived from the cultured isolates of persistently IHA-nonreactive patient sera improved the sensitivity of the IHA. In addition, we assessed the antigen-specific lymphocyte response in this group of patients to a panel of B. pseudomallei antigens, including those derived from their own cultured isolates. Eleven patients with culture-proven melioidosis were identified as having persistently IHA-nonreactive sera. A modified IHA using erythrocytes sensitized with patient isolate-derived antigen tested against convalescent-phase serum was performed. The majority (82%) of sera showed a negative (≤ 1:5) result, one was borderline (1:20), and one was positive at the cutoff value (1:40). IHA-nonreactive sera were also tested by enzyme immunoassay (EIA), with 73% (8/11) demonstrating IgG positivity. In addition, lymphocytes isolated from persistently IHA-nonreactive patient sera demonstrated significantly increased proliferation in response to B. pseudomallei antigens compared to controls. These studies demonstrate the presence of B. pseudomallei-specific antibody by EIA and B. pseudomallei-specific lymphocytes in patient sera categorized as persistently nonreactive according to the IHA. New immunoassays are required and should incorporate B. pseudomallei antigens that are immunoreactive for this subset of IHA-nonreactive patient sera.

Impact of streptozotocin-induced diabetes on functional responses of dendritic cells and macrophages towards Burkholderia pseudomallei.

Diabetes mellitus is a documented risk factor for melioidosis, a tropical infection caused by Burkholderia pseudomallei. The increased susceptibility of diabetic individuals to infections with other pathogens has been associated with immune dysregulation. However, the impact of diabetes on the functional responses of dendritic cells (DC) and macrophages during B. pseudomallei infection has not been investigated. This study compared the responses of macrophages and DC towards B. pseudomallei using bone marrow-derived DC (BMDC) and peritoneal elicited macrophages (PEM) isolated from streptozotocin-induced diabetic C57BL/6 mice exhibiting hyperglycaemia for 9 days (acute) or 70 days (chronic) and age-matched nondiabetic C57BL/6 mice. Following coincubation of BMDC and PEM with a highly virulent B. pseudomallei isolate, maturation, bacterial internalization plus intracellular survival and cytokine gene expression profiles were assessed. No significant differences in functional responses of BMDC or PEM isolated from acute diabetic and nondiabetic mice were observed. However, significant differences in BMDC and PEM function were observed when chronic diabetic and nondiabetic mice were compared. This study demonstrates that diabetic mice with extended periods of uncontrolled hyperglycaemia have impaired DC and macrophage function towards B. pseudomallei, which may contribute to the high susceptibility observed in clinical practice.

Susceptibility to Burkholderia pseudomallei is associated with host immune responses involving tumor necrosis factor receptor-1 (TNFR1) and TNF receptor-2 (TNFR2).

Melioidosis is caused by the facultative intracellular bacterium, Burkholderia pseudomallei. Using C57BL/6 mice, we investigated the role of macrophages, TNF-alpha, TNF receptor-1 (TNFR1) and TNF receptor-2 (TNFR2) in host defense against B. pseudomallei using an experimental model of melioidosis. This study has demonstrated that in vivo depletion of macrophages renders C57BL/6 mice highly susceptible to intranasal infection with B. pseudomallei, with significant mortality occurring within 5 days of infection. Using knockout mice, we have also shown that TNF-alpha and both TNFR1 and TNFR2 are required for optimal control of B. pseudomallei infection. Compared with control mice, increased bacterial loads were demonstrated in spleen and liver of knockout mice at day 2 postinfection, correlating with increased inflammatory infiltrates comprised predominantly of neutrophils and widespread necrosis. Following infection with B. pseudomallei, mortality rates of 85.7%, 70% and 91.7% were observed for mice deficient in TNF-alpha, TNFR1 and TNFR2, respectively. Comparison of survival, bacterial loads and histology indicate that macrophages, TNF-alpha, TNFR1 or TNFR2 play a role in controlling rapid dissemination of B. pseudomallei.

Seropositivity to Burkholderia pseudomallei does not reflect the development of cell-mediated immunity.

Cell-mediated immunity to Burkholderia pseudomallei, the causative agent of melioidosis, provides protection from disease progression. An indirect haemagglutination assay was used to detect antibodies to B. pseudomallei in 1500 healthy donors in an endemic region of Australia. Lymphocyte proliferation, activation and cytokine expression to B. pseudomallei antigen were determined in eight donors who were seropositive and in eight age- and sex-matched controls. In North Queensland, 2.5% of the population was seropositive for B. pseudomallei, which is less than half that which was previously described. Of clinical significance was the observation that while 75% of the seropositive individuals had increased lymphocyte proliferation to B. pseudomallei antigens, there were no significant differences observed in lymphocyte activation or production of cytokines.

Burkholderia pseudomallei enhances maturation of bone marrow-derived dendritic cells.

T-cell activation is essential for protection against Burkholderia pseudomallei infection. Using bone marrow-derived dendritic cells (BMDC) isolated from partially resistant C57BL/6 and susceptible BALB/c mice, the degree of BMDC activation in the presence of B. pseudomallei was investigated. Maturation, cytokine production and internalization of B. pseudomallei by BMDC was assessed in response to infection with a highly virulent and a low-virulent clinical isolate. Maturation was determined by identifying the up-regulation of cell-surface markers CD11c and CD86. IL-1beta and IL-12p40 expression were assessed by reverse-transcriptase PCR. The uptake of B. pseudomallei by BMDC was measured using an internalization assay. This study demonstrated that B. pseudomallei isolates stimulate the maturation of BMDC to the same degree regardless of virulence. However, maturation of BMDC was significantly increased in BALB/c mice compared with C57BL/6 mice. Additionally, the uptake of B. pseudomallei by BMDC was significantly greater with the highly virulent isolate compared with the low-virulent isolate. Expression of IL-12 and IL-1beta following infection with B. pseudomallei was up-regulated. The differences observed may have implications in the development of an effective immune response to B. pseudomallei.